The invention discloses a method for preparing androstenedione (AD) by the whole-cell biotransformation of phytosterol in quaternary phosphonium salt ionic liquid cosolvent system. A mycobacterium sp.NRRL B-3683 strain is cultured by seed solution, the seed solution is then inoculated into fermentation medium and culture to prepare resting cells, the obtained resting cells are activated and cultured in glucose-containing Tris-HCl buffer solution for 7 hours, substrate phytosterol and 0.1 to 1vol.percent of hydrophilic fatty acid quaternary phosphonium salt ionic liquid are poured in as cosolvent, so that biotransformation reaction is carried out, and thereby AD is prepared. The solubility of substrate phytosterol in the hydrophilic fatty acid quaternary phosphonium salt ionic liquid usedby the invention is up to 176g L-1 to 468g L-1, and is increased by more than 100 times in comparison with ordinary commercialized ionic liquids, the dissolution of substrate and product can be effectively promoted, the efficiency of transformation in the process can be increased, and the dosage of ionic liquid is tiny, which helps reduce production cost.