A novel ultra-sensitive molecularly imprinted electrochemical senor with good imprinted capacity to glyphosate was prepared by electropolymerisation on the gold electrode surface,with pyrrole(Py) as functional monomer, glyphosate(Gly) as template. The prepared electrode was characterized by cyclic voltammetry(CV), differential pulse voltammetry(DPV) and electrochemical impedance spectroscopy(EIS). The condition of polymerization,the method of eluting template,the pH value of detection andincubation time were optimized.The optimal conditions were as follows: supporting electrolyte: Britton Robison buffer solution(BR,pH 5.0),ratio of template to functional monomers:1∶5,incubation system:formic acid aqueous solution(pH 4.0),incubation time:18 min.Under the optimum experimental conditions,the prepared electrode showed rapid response, high sensitivity and good selectivity for the template molecule glyphosate. A good linear relationship between oxidation peak current and Gly concentration was obtained over the range of 5-800ng/mL with a correlation coefficient of 0.9817 and a detection limit(S/N=3) of 0.27ng/mL.The prepared sensor also showed good reproducibility and stability, the sensor was successfully applied in the determination of glyphosate in the tap water with recoveries of 78.6%-99.0%.