Abstract: Zebrafish（Danio rerio） has been proven to be a very valuable model animal for studying human tumor biology. However, there is currently a lack of research that combines zebrafish xenografts with single-cell transcriptome analysis. Based on this, therefore, this study first established a zebrafish xenograft model with colon cancer tumor cell line HCT-116. After the tumor cells proliferated in larvae for 4 days, single-cell suspension was obtained by enzymatic hydrolysis and ultra-speed flow cytometry. At the same time, a 4 d single-cell suspension of HCT-116 with synchronized 2D cultured was prepared. Analyzing the single-cell transcriptome data of the above two samples, results of clustering showed that more than 90% of the colon cancer tumor cell line HCT-116 gene expression level in the xenografts was consistent with the synchronized culture in the incubator, indicating that zebrafish as a xenograft host can well maintain the status of tumor cells. But there are still some colon cancer tumor cell lines HCT-116 cells belonging to xenograft models. Differential expression analysis showed that the highly expressed genes of this cluster regulate the process of tumor cells’ proliferation, metastasis and angiogenesis in larvae through multiple pathways. These results provide a new perspective for the systematic evaluation of zebrafish xenograft models.