Bacillus subtilis is an important industrial production strain, widely used in the expression of foreign proteins. At present, among the common constitutive promoters in Bacillus subtilis, PspovG, as a high-strength constitutive promoter, has the advantages of high expression, good stability and wide application when expressing foreign proteins. This study first truncated the upstream sequence of PspovG, and the strength of the promoter was still maintained when the upstream sequence of promoter was shortened form the original 251 bases to 26. And the three bases 'AGC' adjacent to the upstream of element -35 was identified as the key bases affecting the promoter strength. Secondly, random mutations of the upstream A-T rich region of promoter and the G-C rich region of spacer region were conducted, which proved that the arrangement of the A-T bases in the upstream activation sequence had an important influence on the promoter strength, and a mutant with increased strength was obtained. Finally, a novel promoter SPspovG-PlytR with a promoter strength of 135.1% of the original promoter was obtained by inserting the key regulatory sequences of promoters at different expression periods into the upstream and downstream of mutant. This study provides an important reference for the development of new strong constitutive promoters for the Bacillus subtilis expression system.