Recently, people are becoming more concerned about their physical health and putting forward higher requirements for an early and painless diagnosis of diseases. Traditional methods, such as surface plasmon resonance (SPR), enzyme-linked immunosorbent assay (ELISA), surface-enhanced raman spectroscopy (SERS), and colorimetric methods have been used for the detection of biomarkers with high selectivity and sensitivity; however, these methods still need to be further improved for immediate and rapid diagnosis. Herein, organic thin-film transistors (OTFTs)-based biosensors offer the advantages of good flexibility, low-cost fabrication, reasonable sensitivity, and great biocompatibility for efficient determination of biomarkers in complex samples, including saliva, sweat, urine, and blood, respectively, exhibiting great potential in early disease diagnosis and clinical treatment.