An over-current protection module for a flyback power supply having a transformer includes: a switch control unit, configured to generate a control signal in a first period to control a transistor switch coupled to a primary side of the transformer, wherein the transistor switch is turned on in a first duty cycle of the control signal and the transistor switch is turned off in a second duty cycle of the control signal; a transformation unit, configured to generate a compensation current according to the second duty cycle; a timing control unit, configured to output the compensation current to an impedance unit to generate an impedance cross voltage in a shut-off period of the transistor switch of the first period; and a current control unit, configured to determine an over-current reference voltage according to the impedance cross voltage for the over-current protection module in a second period following the first period.