A patient-specific lung cancer assembloid (LCA) model was developed using droplet microfluidic technology to mimic the three-dimensional architecture and heterogeneous tumor microenvironment (TME) of in vivo tumors. This model enables precise manipulation of clinical microsamples and rapid generation of LCAs with good intra-batch consistency in size and cell composition by encapsulating patient tumor-derived TME cells and lung cancer organoids inside microgels. LCAs recapitulate the inter- and intratumoral heterogeneity, TME cellular diversity, and genomic and transcriptomic landscape of their parental tumors. They can reconstruct the functional heterogeneity of cancer-associated fibroblasts (CAFs) and reflect the influence of TME on drug responses compared to cancer organoids. Notably, LCAs accurately replicate clinical outcomes, suggesting their potential for predicting personalized treatments. Lung cancer is the leading cause of cancer deaths, with over 1.8 million deaths worldwide in 2020. Despite advances in therapeutic strategies, few patients achieve complete remission, and responses are highly variable. Tumor heterogeneity and TME contribute to poor treatment outcomes. The TME consists of extracellular matrix and various cellular components, including immune cells and stromal cells. CAFs are major stromal cells in the TME with the ability to drive cancer metastasis and drug resistance. The TME varies greatly between and within patients, posing challenges for precision therapy and drug development. In vitro cancer models have contributed to cancer research and drug development. However, traditional models lack the heterogeneous cell subtypes and molecular features of parental tumors. Patient-derived cancer organoids can replicate the pathological morphology and some genetic features of parental tumors. However, conventional organoid models based on Matrigel mainly represent tumor epithelium, and endogenous stromal and immune cells are gradually lost over time in culture. Although some studies reconstituted a part of the TME in organoid culture systems, some other studies developed non-Matrigel-based hydrogel 3D cancer models, but these models lacked precise controllability and uniformity. Assembloids are 3D structures formed from the fusion and functional integration of multiple cell types or organoids. Bladder tumor assembloids were created and partially recapitulated the in vivo pathophysiological features of urothelial carcinoma. Currently, assembloids are mainly fabricated by coculture and 3D extrusion printing methods. The morphology and structure of assembloids fabricated by coculture methods are difficult to control and have poor intrabatch consistency. Although a kidney organoid model with tissue morphology could be fabricated by using extrusion-based 3D printing, only 18 organoids with diameter of 2 mm could be generated per minute. In this study, an innovative patient-specific lung cancer assembloid (LCA) model was generated by microinjection-based droplet microfluidicA patient-specific lung cancer assembloid (LCA) model was developed using droplet microfluidic technology to mimic the three-dimensional architecture and heterogeneous tumor microenvironment (TME) of in vivo tumors. This model enables precise manipulation of clinical microsamples and rapid generation of LCAs with good intra-batch consistency in size and cell composition by encapsulating patient tumor-derived TME cells and lung cancer organoids inside microgels. LCAs recapitulate the inter- and intratumoral heterogeneity, TME cellular diversity, and genomic and transcriptomic landscape of their parental tumors. They can reconstruct the functional heterogeneity of cancer-associated fibroblasts (CAFs) and reflect the influence of TME on drug responses compared to cancer organoids. Notably, LCAs accurately replicate clinical outcomes, suggesting their potential for predicting personalized treatments. Lung cancer is the leading cause of cancer deaths, with over 1.8 million deaths worldwide in 2020. Despite advances in therapeutic strategies, few patients achieve complete remission, and responses are highly variable. Tumor heterogeneity and TME contribute to poor treatment outcomes. The TME consists of extracellular matrix and various cellular components, including immune cells and stromal cells. CAFs are major stromal cells in the TME with the ability to drive cancer metastasis and drug resistance. The TME varies greatly between and within patients, posing challenges for precision therapy and drug development. In vitro cancer models have contributed to cancer research and drug development. However, traditional models lack the heterogeneous cell subtypes and molecular features of parental tumors. Patient-derived cancer organoids can replicate the pathological morphology and some genetic features of parental tumors. However, conventional organoid models based on Matrigel mainly represent tumor epithelium, and endogenous stromal and immune cells are gradually lost over time in culture. Although some studies reconstituted a part of the TME in organoid culture systems, some other studies developed non-Matrigel-based hydrogel 3D cancer models, but these models lacked precise controllability and uniformity. Assembloids are 3D structures formed from the fusion and functional integration of multiple cell types or organoids. Bladder tumor assembloids were created and partially recapitulated the in vivo pathophysiological features of urothelial carcinoma. Currently, assembloids are mainly fabricated by coculture and 3D extrusion printing methods. The morphology and structure of assembloids fabricated by coculture methods are difficult to control and have poor intrabatch consistency. Although a kidney organoid model with tissue morphology could be fabricated by using extrusion-based 3D printing, only 18 organoids with diameter of 2 mm could be generated per minute. In this study, an innovative patient-specific lung cancer assembloid (LCA) model was generated by microinjection-based droplet microfluidic