The work aims to explore preparation method of EPS composite coating, and verify its anti-corrosion effect. Based upon electrostatic adsorption among nano-silica and poly (allylamine hydrochloride, PAH) and entrapping extracellular polymeric substances (EPS), a stable and uniform composite coating was prepared by introducing Layer-by-Layer (LbL) self-assembly technology. Stability and anti-corrosion effect of the coating were analyzed by means of Zeta potential, average grain diameter, corrosion weight loss experiment, electrochemical testing, X-ray diffractometer (XRD) and scanning electron microscope (SEM). Zeta potential, average grain diameter test of nano-silica solution, corrosion weight loss experiment and electrochemical experiment indicated that, the coating had the most efficient, stable and lasting anti-corrosion effect provided with mass percent of 1.5wt%, alcohol fraction of 30%, EPS solution concentration of 200 mg/L and layer number of n=7. Scanning electron microscope (SEM), XRD and electrochemical impedance spectroscopy (EIS) results showed that carbon steel coated with LbL multi-layer coating had more compact and orderly corrosion products (quantities of Fe3O4) after being exposed to corrosive conditions for 15 days, lowest corrosive electric current density and maximum impedance. Optimum parameters and preparation method of LbL-based EPS composite coating are obtained. Compared with the original carbon steel and EPS coating, the anti-corrosion effect of the multi-layer EPS coating coated with LbL is the best.