A biosensor, and a preparation and biosensing method therefor. The biosensor includes: a sensing substrate, wherein a plurality of sensing suspending arms arranged in an array are arranged on the sensing substrate, and the sensing suspending arms have identification markers; and a detection substrate, the detection substrate including a plurality of light detection assemblies arranged in an array, wherein the light detection assemblies and the sensing suspending arms are arranged in one-to-one correspondence, each of the light detection assemblies includes a photodiode and a thin film transistor, and the photodiode is connected to the thin film transistor.