The Gal4/UAS system provides a powerful tool to analyze the function of genes. The system has been employed extensively in zebrafish; however, cytotoxicity of Gal4 and methylation of UAS can hinder future applications of Gal4/UAS in zebrafish. In this study, we provide quantitative data on the cytotoxicity of Gal4-FF and KalTA4 in zebrafish embryos. A better balance between induction efficiency and toxicity was shown when the injection dosage was 20pg for Gal4-FF and 30pg for KalTA4. We tested the DNA methylation of UAS in different copies (3x, 5x, 7x, 9x, 11x, and 14x), and the results showed, for the first time, that the degree of UAS methylation increases with the increase in the copy number of UAS. We detected insertions of the Tol2-mediated transgene in the Gal4 line and found as many as three sites of insertion, on average; only about 20% of individuals contained single-site insertion in F1 generation. We suggested that the screening of Gal4 lines with single-site insertion is essential when Tol2-mediated Gal4 transgenic lines are created. Moreover, we designed a novel 5xnon-repetitive UAS (5xnrUAS) to reduce the appeal of multicopy UAS as a target for methylation. Excitingly, the 5xnrUAS is less prone to methylation compared to 5xUAS. We hope the results will facilitate the future application of the Gal4/UAS system in zebrafish research.