Objective To investigate the best digestion method for the preparation of single cell suspensions from mouse small intestinal lamina propria. Methods Ten mouse small intestines of uniform length were collected and randomly divided into two groups. Each group prepared the lamina propria single cell suspensions by enzymatic digestion with collagenase A or collagenase VIII, and compared the effects of these two enzymatic digestion methods on the cell yield, cell activity and cell surface antigen of the single cell suspensions, and then the single cell suspensions prepared by the superior enzymatic digestion method were subjected to flow assay. Results Compared to collagenase VIII-based enzymatic digestion, collagenase A-based enzymatic digestion resulted in higher cell yields (9.48 ?1.10) ?109 vs. (4.18 ?1.02) ?109, live cell proportions (86.36 ?3.32) % vs. (61.62 ?10.93) % and active CD45 cells (57.19 ?5.11) ) % vs (26.01?1.44) %, active CD3 cells (8.73?.89) % vs (4.52?.49) %, active CD4 cells (6.19?.09) % vs (3.22?.91) % and active B220 cells (15.06?.27) % vs (5.07?.20) %, providing high quality cells for subsequent flow assays. Conclusion The collagenase A-based enzymatic digestion method is more suitable for the preparation of single cell suspensions from the lamina propria of the mouse small intestine.