A detection substrate and a manufacturing method thereof, and a detector are provided. The detection substrate comprises a base substrate, a thin film transistor, a PIN photodiode and a scintillation layer. The thin film transistor and the PIN photodiode are provided above a first face of the base substrate and the scintillation layer is provided above a second face of the base substrate. The visible light obtained after the X-ray passes through the scintillation layer is directly irradiated on the PIN photodiode after passing through the base substrate with relative high transmittance, thus preventing intensity of the light irradiated on the PIN photodiode from being weakened, and improving light utilization efficiency of the detection substrate.