Transgenesis, which inserts exogenous DNA into animal genomes, is a widely used technique. Traditionally, the constructs for transgenesis are generated by step-by-step subcloning of DNA fragments, which requires multiple steps depending on the construct complexity. To overcome the limitation, advanced tools such as Gateway cloning (Hartley et al., 2000; Kwan et al., 2007), In-Fusion cloning (Sleight et al., 2010), and Gibson assembly (Gibson et al., 2009) have been developed. However, due to their ligation characteristics, no systematic method for transgenesis has been developed. ‘Golden Gate’ cloning first appeared in 2008, which is a widely used DNA assembly method (Engler et al., 2008, 2009). Here, we take zebrafish transgenesis as an example and develop a standardized system called GoldenFish, which is based on Golden Gate cloning. It can customize transgenic constructs in one step and can be applied to multiple types of transgenesis such as one gene driven by one promoter, multiple genes driven by one promoter, and multiple genes respectively driven by multiple promoters, significantly reducing working time.