The invention discloses a pest and glyphosate resistant expression vector, and a plasmid and an application thereof. The expression vector contains a pest-resistant gene cry1Ab, a pest-resistant gene cry2Ab and a glyphosate-resistant gene G10evo. The vector simultaneously expresses Cry1Ab, Cry2Ab and G10evo proteins, and transgenic crops obtained by transferring crops with the vector can simultaneously resist cotton bollworms, beet armyworms, corn borers, striped rice borer and other main lepidoptera pests of rice, corn and cotton, so the insecticidal spectrum is widened, the pest resistance is effectively delayed, the transgenic crops have glyphosate resistance, the transgenic crops with composite properties accord with the current development direction of transgenic crops, and demands of large-scale agricultural production are met.