Disclosed are a method for biological total synthesis of paclitaxel key precursor substance baccatin III, a biological material and use thereof. The bioenzyme composition used in the method comprises a taxane 9α-hydroxylase (T9αH) and a taxane C4-C20 oxetanase (TOT). According to the disclosure, a core component of a baccatin III biosynthetic pathway is identified, a baccatin III artificial synthesis route is constructed, thereby paving the way for efficiently developing a green low-carbon production pathway of taxane products (such as paclitaxel) through synthetic biology.