This study presents an acoustofluidic system for producing structured cell-laden hydrogel fibers/tubules with tunable cell patterns. The system uses acoustic cell patterning, which offers biocompatibility, wide tunability, and contact-free manipulation. Cells or microparticles are pre-patterned in a liquid hydrogel before being extruded as cross-linked hydrogel fibers/tubules. The radial patterns can be tuned based on acoustic resonances, enabling the formation of cell or microparticle pre-patterns. The cell patterns are adjustable based on acoustic frequencies, allowing for the creation of 2/4/6-petal radiant cell assemblies. Cell viability assays after 72 h confirm good cell viability and proliferation. The method is biocompatible and reliable, making it suitable for various biomimetic fabrications. The system consists of a glass capillary, a piezoelectric transducer, and a UV light source. The pre-cross-linked cell-hydrogel suspension is injected into the capillary, where acoustic pressure fields enable cell patterning. The UV light cross-links the hydrogel, maintaining the cell patterns to form structured cell-laden fibers/tubules. The system can produce hydrogel fibers and tubules with patterned particles or cells, demonstrating the ability to create biomimetic structures. The method is suitable for bioprinting and can be integrated into 3D bioprinting systems to create biocompatible scaffolds with controlled architecture and cell distribution. The study shows that the method is effective for creating structured cell-laden hydrogel fibers/tubules with tunable cell patterns, which has potential applications in tissue engineering and regenerative medicine.This study presents an acoustofluidic system for producing structured cell-laden hydrogel fibers/tubules with tunable cell patterns. The system uses acoustic cell patterning, which offers biocompatibility, wide tunability, and contact-free manipulation. Cells or microparticles are pre-patterned in a liquid hydrogel before being extruded as cross-linked hydrogel fibers/tubules. The radial patterns can be tuned based on acoustic resonances, enabling the formation of cell or microparticle pre-patterns. The cell patterns are adjustable based on acoustic frequencies, allowing for the creation of 2/4/6-petal radiant cell assemblies. Cell viability assays after 72 h confirm good cell viability and proliferation. The method is biocompatible and reliable, making it suitable for various biomimetic fabrications. The system consists of a glass capillary, a piezoelectric transducer, and a UV light source. The pre-cross-linked cell-hydrogel suspension is injected into the capillary, where acoustic pressure fields enable cell patterning. The UV light cross-links the hydrogel, maintaining the cell patterns to form structured cell-laden fibers/tubules. The system can produce hydrogel fibers and tubules with patterned particles or cells, demonstrating the ability to create biomimetic structures. The method is suitable for bioprinting and can be integrated into 3D bioprinting systems to create biocompatible scaffolds with controlled architecture and cell distribution. The study shows that the method is effective for creating structured cell-laden hydrogel fibers/tubules with tunable cell patterns, which has potential applications in tissue engineering and regenerative medicine.