Abstract: To increase antibiotic production, the rifampicin resistant was conferring on a Strptomyces Cavourensis strain which was screened in the early. Ribosome engineering technology was used to enable the strain to get rifampicin resistance by ropB gene mutation of strain TJ430. Positive and negative mutant strains was screened by high throughput screening model combined with HPLC. Liquid fermentation was used to further confirm the preliminary screening results. The mutation site of ropB gene was confirmed by gene sequencing. A high throughput screening model was constructed to screen the mutant strains accurately and rapidly. Five positive and two negative mutant strains were isolated. A point mutation was found in the 280bp of the negative mutant ropB gene, but no mutation was found in the sequenced fragment of the positive mutant. The partial sequence of ropB gene can be sequenced by the primer sequence. Compared with the original strain, the yield of the strain was increased more than two and a half times, and the morphological changes of the hyphae were greatly changed thus it can be seen, the mutation site of the positive mutation strain should be located in the other fragment of ropB gene which was not sequenced. Based on the whole genome sequencing of the original strain, the whole ropB gene of the strain could be sequenced by redesigning primers to search for the mutation site.