scCRISPR-seq is an emerging high-throughput CRISPR screening technology that combines CIRPSR screening with single-cell sequencing technologies. It provides rich information on gene regulation. When performing scCRISPR-seq in a population of heterogeneous cells, the observed cellular response in perturbed cells may be caused not only by the perturbation, but also by the infection bias of guide RNAs (gRNAs) mainly contributed by intrinsic differences of cell clusters. The mixing of these effects poisons gene regulation studies. We developed scDecouple to decouple the true cellular response of the perturbation from the influence of infection bias. It models the distribution of perturbed cells and iteratively finds the maximum likelihood of cell cluster proportions as well as the real cellular response for each gRNA. We demonstrated its performance on a series of simulation experiments. By applying scDecouple to real CROP-seq data, we found that scDecouple could enhance biological discovery by detecting perturbation-related genes more critically. It helps to better study gene function and identify disease targets via scCRISPR-seq, especially with heterogeneous samples or complex gRNA libraries.