〔Abstract〕 Objective To compare the biological behavior between rhabdomyosarcoma(RMS) fusion gene positive and negative cells, analyze differential miRNAs of RMS fusion gene positive and negative using bioinformatics, and verify the expression of differential miRNAs. Methods The viability, invasion, migration and apoptotic capacity of fusion gene positive RH30 cells and fusion gene negative RD and PLA-802 cells in RMS were detected by CCK-8, Transwell, and flow cytometry, respectively. GEO2 R was used to analyze the differential miRNA of the fusion gene positive and negative cells. Sangerbox was used to analyze differential miRNAs and made Volcano map. qRT-PCR was used to verify the selected differential miRNAs. Results The proliferation ability of RD cells was higher than that of RH30 cells and PLA-802 cells(P<0.05). The invasion, migration and anti-apoptotic ability of RH30 cells were higher than those of PLA-802 cells and RD cells(P<0.05). Volcano plot results showed that the number of up-regulated miRNAs was more than down-regulated miRNAs(P<0.05). According to |log2 FC|>3,P<0.05, 12 differential miRNAs were screened(up-regulated miRNAs contained miR-1, let-7 d-5 p and let-7 b-5 p, down-regulated miRNAs contained miR-196 a-5 p, miR-455-3 p, miR-21-5 p, miR-193 a-3 p, miR-29 b-3 p, miR-29 a-3 p, miR-100-5 p, miR-222-3 p and miR-221-3 p), and qRT-PCR results were consistent with bioinformatics analysis. Conclusion The ability of invasion, migration and resistant apoptosis in RMS fusion gene-positive cells is higher than that of fusion gene-negative cells. The differentially expressed miRNAs between fusion gene positive and negative groups can provide new directions for RMS research.