The cylinder plate method for determination of bacitracin residues in aquatic products (grass carp,eel and prawn muscle) was established. The samples were extracted with methanol and 0.1% formic acid water (volume ratio 3∶7) under ultrasonication,reduced pressure distillation and then using Micrococcus luteus to detect residual after the concentration of samples. The results showed that the detection limit of bacitracin was 0.25 μg/g in grass carp muscle and 0.50 μg/g in eel and prawn muscle,which satisfied the maximum residue standards of muscle tissue ruled by Chinese Ministry of Agriculture,as well as the European Union. When bacitracin in the muscle tissue of aquatic products ranges from 1 to 10 μg/mL,it showed a good linearity between drug concentration logarithm and the diameter of bacteriostatic (R2>0.990 0). For different aquatic products,when additive amount of bacitracin ranged from 0.5 μg/g to 2.5 μg/g,the recoveries of all muscle tissues were 70.45%-93.44%,and the relative standard deviations were 2.02%-12.62%. Furthermore,three different bacterial test plates was adapted to ensure the residues in aquatic products is bacirtracin.