Background: The tubers of Dioscorea zingiberensis, is the most favorable plant material for the production of diosgenin, an important bioactive steroidal sapogenin and requisite precursor of cortin, contraceptive and sex hormone, which is the only desired product after steroidal saponins from the tubers are hydrolyzed. Objective: A novel technology, in situ pressurized biphase acid hydrolysis was constructed for the first time to simplify extraction process, increase extraction yield and decrease the consumption of mineral acids. Materials and Methods: The method developed in this study has been optimized and verified through orthogonal design for experiments, in which the effect and their significance of four factors including molarity of acid, temperature, extraction duration and sample quantity have been investigated. Then, the comparison was conducted among the newly developed method and other reported methods. The diosgenin was also isolated by column chromatography, followed by mass spectrometry and nuclear magnetic resonance analysis for structural confirmation. Results: It was found that temperature is the factor of the most influence and the highest extraction yield at 2.21% has been achieved while the hydrolysis was performed at 140°C for 1.5 h in 0.20M H2SO4 solution with petroleum ether under an uncontrolled pressurized condition. And, compared to the others, the increment in the extraction yield of new method was 20.8 ~ 74.0%, and the consumption of H2SO4 was reduced by 17 times at most. Conclusion: This method is a much cleaner and more efficient approach for extraction of diosgenin from the tubers, and is promising to be applied in pharmaceutical industry.