A single-molecule enzyme-linked immunosorbent assay (digital ELISA) enables the detection of serum proteins at subfemtomolar concentrations. This method uses arrays of femtoliter-sized reaction chambers (SiMoA) to isolate and detect single enzyme-labeled proteins. Proteins are captured on microscopic beads and labeled with an enzyme, allowing for the detection of very low concentrations of proteins. By isolating these beads in arrays of 50-femtoliter reaction chambers, single proteins can be detected by fluorescence imaging. Digital ELISA detected prostate-specific antigen (PSA) in all tested sera from patients who had undergone radical prostatectomy, down to 14 fg/mL (0.4 fM). The method allows for the detection of clinically-relevant proteins in serum at concentrations much lower than conventional ELISA. The high sensitivity of SiMoA to enzyme labels, combined with the low background signals achieved by digitizing the detection of proteins, enables the measurement of clinically important proteins in serum at sub-femtomolar concentrations. This approach has the potential to facilitate the early diagnosis and treatment of disease by enabling the detection of very low concentrations of proteins in blood. The method was validated using clinically-relevant proteins, including PSA and tumor necrosis factor-α (TNF-α), and demonstrated the ability to detect these proteins at sub-femtomolar concentrations. The results show that digital ELISA provides a significant improvement in sensitivity compared to conventional ELISA, with the potential for diagnostic benefits in clinical applications.A single-molecule enzyme-linked immunosorbent assay (digital ELISA) enables the detection of serum proteins at subfemtomolar concentrations. This method uses arrays of femtoliter-sized reaction chambers (SiMoA) to isolate and detect single enzyme-labeled proteins. Proteins are captured on microscopic beads and labeled with an enzyme, allowing for the detection of very low concentrations of proteins. By isolating these beads in arrays of 50-femtoliter reaction chambers, single proteins can be detected by fluorescence imaging. Digital ELISA detected prostate-specific antigen (PSA) in all tested sera from patients who had undergone radical prostatectomy, down to 14 fg/mL (0.4 fM). The method allows for the detection of clinically-relevant proteins in serum at concentrations much lower than conventional ELISA. The high sensitivity of SiMoA to enzyme labels, combined with the low background signals achieved by digitizing the detection of proteins, enables the measurement of clinically important proteins in serum at sub-femtomolar concentrations. This approach has the potential to facilitate the early diagnosis and treatment of disease by enabling the detection of very low concentrations of proteins in blood. The method was validated using clinically-relevant proteins, including PSA and tumor necrosis factor-α (TNF-α), and demonstrated the ability to detect these proteins at sub-femtomolar concentrations. The results show that digital ELISA provides a significant improvement in sensitivity compared to conventional ELISA, with the potential for diagnostic benefits in clinical applications.