To analyze the best conditions for different types of mutations required in error-prone PCR system, this paper tried to process error-prone PCR by adding different concentrations of manganese(0.125，0.250 and 0.500 mmol/L) into normal PCR system. Different lengths of DNA fragments of S.cerevisiae spt15 (about 200,500 and 700 bp) were amplified to study their mutation ratios, spectrum of mutations and the distribution of base substitutions. The results have shown that the error-prone PCR mutation rate of longer fragment DNA is more sensitive to manganese ions concentration. The mutation number of bases has a significant impact on the length of template DNA. Lower manganese ions concentration is conducive to a single mutation. The base substitution distribution of mutation driven by manganese ions has obvious bias towards the transition of A → G and T → C.