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This invention belongs to the field of microbial pesticides and discloses a highly effective Bacillus subtilis strain for killing spider mites and its applications. The Bacillus subtilis strain described in this invention has the preservation number CCTCC NO: M 20251959. Indoor bioassay data show that Bacillus subtilis NBIF-051 has high toxicity against major agricultural mites such as the two-spotted spider mite, the citrus red spider mite, and the cotton red spider mite. Pot experiments showed that after spraying the fermented broth of NBIF-051 for 7 days, the control efficacy against the two-spotted spider mite was 65.18%. Field trials showed that after spraying the fermented broth of this strain for 15 days, the control efficacy against the citrus red spider mite was 63.45%~76.85%, comparable to commercially available chemical acaricides. Furthermore, the acaricidal activity of the fermented broth of this strain exhibits certain tolerance to high temperatures and ultraviolet radiation, showing broad application prospects in agricultural production.