Hainan is the dominant production area of the red-fleshed pitaya (Hylocereus undatus) cv. ‘Jindu No.1’ in China, and cutting propagation is the main method for its large-scale seedling cultivation. Plant growth regulators (PGRs) are the key factors regulating the rooting of cuttings. Existing studies mostly focus on the concentration optimization of a single agent, lack systematic broad-spectrum screening of commonly used PGRs in agriculture, and have the problem of disconnection between laboratory results and field production. To screen an efficient root-promoting PGR scheme suitable for large-scale seedling cultivation in Hainan production areas, this study established a three-level experimental system of “broad-spectrum primary screening→gradient re-screening→soil culture scenario verification”, used 14 kinds of PGRs commonly used in agricultural production as materials, and carried out a systematic evaluation combined with principal component analysis (PCA). 1-naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA) and potassium indole-3-butyrate (K-IBA) were identified as high-efficiency agents in the primary screening, with a rooting rate of 100%, and the core root morphological indexes were significantly better than those of the water control (p < 0.05). Two independent experiments verified the stability of the “total growth–thickness” binary regulation mechanism of the pitaya root system. In the re-screening test, 400 mg·L⁻¹ NAA had the best comprehensive performance, synergistically improving the total root growth and root thickness, and 125 mg·L⁻¹ K-IBA had the most significant effect in promoting the longitudinal extension of roots, with the average root length increased by 760.0% compared with the control. Soil culture tests confirmed that the two optimal schemes had stable and reliable application effects in field substrate cultivation. The results of this study can provide technical support for the large-scale seedling cultivation of ‘Jindu No.1’ pitaya, and the established three-level screening system also provides a methodological reference for PGR screening in cutting propagation of similar tropical crops.