Targeted gene replacement via homologous recombination (HR) is a conventional approach for the analysis of gene function. However, this event is rare in Beauveria bassiana, which hampers efficient functional analysis in this widely used entomopathogenic fungus. To improve homologous recombination frequency in B. bassiana, we investigated the effect of the ratio of homologous sequence to non-homologous sequence (HS/NHS) in gene disruption cassette upon the HR frequency by two gene loci BbNtl and BbThi, using the herpes simplex virus thymidine kinase as a negative selectable marker against ectopic transformants. Our data revealed that an increase of the ratio of HS/NHS achieved by either extending homologous sequence or decreasing non-homologous sequence could improve HR frequency in B. bassiana. We determined empirically that (1) at least 700 bp of homology to both sides of a target gene was needed to get a reasonable number of disruptants, e.g., 6.7 per thousand to 13.3 per thousand in B. bassiana. (2) When the ratio of HS/NHS was above 0.8, an acceptable HR frequency could be achieved for gene replacement in B. bassiana, while when the ratio was below 0.3, few gene disrupted mutants were obtained.