Objective The stimulus color of P300-BCI systems has been successfully modified. However, the effects of different color combinations have not been widely investigated. In this study, we designed new stimulus patterns to evaluate the influence of color modulation on the BCI performance and waveforms of the evoked related potential (ERP).Methods Comparison was performed for three new stimulus patterns consisting of red face and colored block-shape, namely, red face with a white rectangle (RFW), red face with a blue rectangle (RFB), and red face with a red rectangle (RFR). Bayesian linear discriminant analysis (BLDA) was used to construct the individual classifier model. Repeated-measures ANOVA and Bonferroni correction were applied for statistical analysis. Results The RFW pattern obtained the highest average online accuracy with 96.94%, and those of RFR and RFB patterns were 93.61% and of 92.22% respectively. Significant differences in online accuracy and information transfer rate (ITR) were found between RFW and RFR patterns (p < 0.05). Conclusion Compared with RFR and RFB patterns, RFW yielded the best performance in P300-BCI. These new stimulus patterns with different color combinations have considerable importance to BCI applications and user-friendliness.