The eznyme biosensor(EB) as a sort of biosensors are widely used analyse target substances from samples of medicine, environment and food processing in recent years. During the EB preparation, effective immobilization of enzyme is a key step related with the activity of enzyme and the lifetime of EB. In current research, a method to determine nonylphenol(NP) contents in the water samlples has been developed on the base of laccases cetyltrimethyl ammonium bromide -carbon paste electrode. The experimental results showed that the EB developed in this optimized conditions, its minimum detectable limit could reach 0.2µg/L, and the recovery was in the range from 91% to 109% at the concentrations form 3.0×10-4 mol.L-1 to 5.0×10-6 mol.L-1. The sensor is perfect which is convenient, effective, and cheap and really can be used practice.