Objective To clone the encoding gene of surface layer (S-layer) protein of Lactobacillus acidophilus, realize the prokaryotic expression, and purify expression products. Methods The gene of S-layer protein of Lactobacillus acidophilus was extracted, amplified, ligated to the expression vector of pET-SUMO3, and transcribed and expressed in E.coli BL21 (DE3). The S-layer protein was extracted and purified by the nickel-affinity chromatography. The expression products were verified by the SDS polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Results The encoding gene of S-layer protein of Lactobacillus acidophilus ATCC4356 was cloned and had 98% homology with the relevant gene. Results of the SDS-PAGE and Western blotting showed that the fusion protein expressed efficiently in bacteria. The recombinant S-layer protein (43 000 Da) could be obtained by the affinity chromatography and removing the fusion tag by the enzymatic digestion. Conclusion The prokaryotic expression can be achieved and the S-layer protein with high purity can be acquired by obtaining the encoding gene of S-layer protein of Lactobacillus acidophilus. These are helpful for subsequent studies on biological functions.