[Background] Staphylococcus aureus is an important pathogen, and procoagulation is one of the most important pathogenic mechanism, possibly novel genes in this approach. [Objective] To screen and identify procoagulant genes of Staphylococcus aureus by observing the decreased agglutination of colony phenotype. [Methods] Random mutagenesis library was constructed by using Tn917 transposon system, and mutants would be screened by combining dynamic turbidimetry and tube agglutination. The related genes were identified, and putative function of these genes was predicted through bioinformatics analysis. [Results] A total of 82 mutants were selected in a way of decreased agglutination phenotypes, and insertion sites of 76 mutants were successfully identified with 13 mutated genes, including 4 were reported in previous studies (which is 30.8% of these genes identified). [Conclusion] We acquired related genes based on decreased agglutination of mutants, and this was a new method of identifying potential genes involved in procoagulant ability, at the same time, new agglutination candidates obtained were reserved for further study.