Red pitaya, the representative tropical and subtropical fruit, is vulnerable to quality deterioration due to climate or agronomic measures. Nano-selenium (Nano-Se) has shown positive effects on crop biofortification in favour of reversing this situation. In this study, Se could be enriched efficiently in red pitayas via root and foliar application by Nano-Se, which induced higher phenolic acids (16.9-94.2%), total phenols (15.7%), total flavonoids (29.5%) and betacyanins (34.1%) accumulation in flesh. Richer antioxidative features including activities of SOD (25.2%), CAT (33.8%), POD (77.2%), and levels of AsA (25.7%) and DPPH (14.7%) were obtained in Nano-Se-treated pitayas as well as in their 4-8 days shelf-life. The non-targeted metabolomics indicated a boost in amino acids, resulting in the stimulation of phenylpropanoid and betalain biosynthesis. In conclusion, the mechanism of Nano-Se biofortification for red pitaya might be fortifying pigment, as well as the enzymatic and non-enzymatic antioxidant substances formation by regulating primary and secondary metabolism facilitated by Se accumulation.