Objective To investigate the optimal parameters of the ultrasound combined with microbubbles promoting SDF-1 expression in human mesenchymal stem cells according to the quasi-level uniform design. Methods The acoustic intensity, irradiation time, and the dosage of MBs were selected, then five levels of each factor were chosen, acoustic intensity (0 W/cm2、0.2 W/cm2、0.4 W/cm2、0.6 W/cm2、0.8 W/cm2)，irradiation time (0 s、20 s、30 s、40 s、60 s), and the dosage of MBs (0 N/ml、102 N/ml、104 N/ml、106 N/ml、108 N/ml). The uniform design table U15(155) and the using tables were set, and the rat mesenchymal stem cells were radiated according to the uniform experimental design. After ultrasound radiation, the cells were cultured for 24 h, then the concentration of SDF-1 in cultural supernatant and the cell viability were detected. Following the principle of more SDF-1 secretion and less cell death, the optimized parameters were determined by regression analysis and then validated by orthogonal experimental. Results Considering good cell viability, the optimal conditions for SDF-1 secretion were acoustic intensity of 0.6 W/cm2 , irradiation time of 30 s and the dosage of MBs of 106 N/ml. The results of verification experiments showed that after microbubble mediated ultrasound irradiation, the concentration of SDF-1 was 551.67±40.88 pg/ml and the cell viability was 88.51±4.03% by using the optimized parameters. Conclusion The application of quasi-level uniform design can effectively optimize the parameters of microbubble mediated ultrasound irradiation, and find a balance between SDF-1 secretion and cell viability of mesenchymal stem cells.