The invention relate to a shuttle expression BAC vector among colon bacillus, streptomyces and pseudomonad, and a construction method thereof. The BAC vector contains a replicon of pUC18, a penbritin resistance gene, a conjugal transfer fragment oriT, an attP locus, an integrase gene int, an Am resistance gene, a 5' end of a PP_0423' gene of the pseudomonad, a crp gene of the pseudomonad, a redF gene of the BAT vector, duplicate field OriS, a repA gene, a sopA gene, a sopB gene, a sopC gene, a cos locus, and a loxP locus. The method starts from pECBAC1, effectively combines and optimizes a necessary original of heterologous expression, applies means such as gene clone and recombination engineering and so on, successfully constructs the shuttle expression BAC vector, and provides the convenience for biosynthetic gene clusters with the heterologous expression and high throughput screening.