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Organic field-effect transistors (OFETs) are fabricated using organic semiconductors (OSCs) as the active layer in the form of thin films. Due to its advantages of high sensitivity, low cost, compact integration, flexibility, and printability, OFETs have been used extensively in the sensing area. For analysis platforms, the construction of sensing layers is a key element for their efficient detection capability. The strategy used to immobilize biomolecules in these devices is especially important for ensuring that the sensing functions of the OFET are effective. Generally, analysis platforms are developed by modifying the gate/electrolyte or OSC/electrolyte interface using biomolecules, such as enzymes, antibodies, or deoxyribonucleic acid (DNA) to ensure high selectivity. To provide better or more convenient biological immobilization methods for researchers in this field and thereby improve detection sensitivity, this review summarizes recent developments in the immobilization strategies used for biological macromolecules in OFETs, including cross-linking, physical adsorption, embedding, and chemical covalent binding. The influences of biomolecules on device performance are also discussed.