The invention discloses a preparation method of a 96-well enzyme label plate AOZ molecularly imprinted polymer film. The preparation method comprises the following steps of a, dissolving AOZ as a template molecule, 2-vinylpyddine as a functional monomer and ethylene glycol dimethacrylate as a cross-linking agent in ethanol as a pore forming agent according to a mole ratio of 1: 4: 1, and carrying out ultrasonic treatment for 30min, wherein a use amount ratio of ethanol to AOZ is 5ml/0.25mmol, b, adding azodiisobutyronitrile as an initiator into the mixture, and carrying out ultrasonic treatment for 15min, wherein a use amount ratio of azodiisobutyronitrile to AOZ is 25mg/0.25mmol, c, dropwisely adding the solution obtained by the step b on a 96-well enzyme label plate, putting the 96-well enzyme label plate into a sealing bag, filling nitrogen into the sealing bag, carrying out sealing, and carrying out ultraviolet lamp irradiation to initiate polymerization lasting for 24h, d, putting the 96-well enzyme label plate into a methanol-acetic acid mixed solution having a volume ratio of 3: 1, and carrying out ultrasonic elution for 48h, and e, drying the 96-well enzyme label plate for 2h at a temperature of 50 DEG C to obtain the AOZ molecularly imprinted polymer film.