Objective To investigate the optimal 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 was used to prepare lamina propria single-cell suspensions by enzymatic digestion with collagenase A or collagenase VIII. We compared the effects of these two enzymatic digestion method on the cell yield, cell activity, and cell surface antigens of the single-cell suspensions. Single-cell suspensions prepared by the superior enzymatic digestion method were then subjected to flow cytometry assay. Results Compared to collagenase-VIIIbased enzymatic digestion, collagenase-A-based digestion result ed in a higher cell yield (9. 48±1. 10) ×10⁹ vs (4. 18±1.02)×10⁹ and higher proportions of live cells ( 86. 36± 3. 32)% vs ( 61. 62± 10. 93)%, active CD45 ⁺ cells ( 57. 19±5. 11)% vs ( 26. 01 ± 11. 44)%, active CD3 ⁺ cells ( 8. 73 ± 2. 89)% vs ( 4. 52 ± 2. 49)%, active CD4 ⁺ cells ( 6. 19 ±2. 09)% vs (3. 22±1. 91)%, and active B220 ⁺ cells (15. 06±4. 27)% vs (5. 07±2. 20)%, providing high-quality cells for subsequent flow assays. Conclusions The collagenase A-based enzymatic digestion method is more suitable for the preparation of single-cell suspensions from the lamina propria of mouse small intestines.