Abstract Selenium (Se) is an essential trace element that plays a critical role in human tissue formation, metabolism, and physiological functions. However, many individuals worldwide suffer from Se deficiency diseases. This study aims to evaluate the impact of Se-tolerant LF-17 agents and exogenous Na2SeO3 application on the growth, enzyme activity, and metabolic characteristics of rape seedlings. Treatment LF-3 (inoculation of Se-tolerant LF-17 agent and exogenous Na2SeO3, with the soil Se concentration of 5 mg/kg) led to a 38.62% increase in plant height and a 116.7% increase in fresh weight. And the Se-tolerant LF-17 agent in treatment LF-3 also reduced the oxidative stress induced by exogenous Na2SeO3 compared to that of treatment LF-2 (with the same amount exogenous Na2SeO3 only), as evidenced by the lower activities of SOD, POD, and CAT, as well as less content of malondialdehyde. Furthermore, the upregulation of metabolic pathways such as “cuticle, suberine, and wax biosynthesis” “flavonoid biosynthesis,” and “terpenoid backbone biosynthesis” enhanced the plant’s stress resistance as revealed by non-targeted metabolomics sequencing method. This approach offers promising applications for improving Se bioavailability in crops, mitigating Se toxicity, addressing global Se deficiency challenges and is expected to contribute to fulfilling the Se supplementation needs of the population.