Steroids exhibit a range of biological activities and are commonly described as the ‘key to life’ in nature. Steroidal-based medications have emerged as the second largest pharmaceutical category following antibiotics, owing to their remarkable bioactivities such as anti-infective, anti-inflammatory, anti-allergic, and antitumor properties. This category encompasses more than 400 drug compounds, representing approximately 17% of FDA-approved medications. The synthesis of steroidal products continues to attract significant attention due to their diverse bioactivities and physicochemical characteristics in pharmaceutical applications. With the increasing demand for steroidal drugs and the fluctuating availability of sapogenin resources, the use of Mycobacteria to convert inexpensive phytosterols to produce key intermediates for steroid drugs has been established as the most mature and sustainable industrial route. However, the complex structure of steroids, particularly their highly oxygenated skeleton, poses challenges for the well-established semi-synthesis route of complex steroid medications. Recent strides in bioinformatics and genetics have significantly advanced the studies on synthesis of steroidal compounds. This review highlights recent advancements in the synthesis of high-value steroids, including the diverse steroid drug intermediate production via external steroidal modifying enzymes expression in engineered Mycobacteria, chemo-enzymatic synthesis of complex steroids, and yeast-based de novo synthesis. It specifically highlights the significant achievements in the chemo-enzymatic synthesis, which combines the precise site- and stereoselectivity of enzymatic transformations with the efficiency of chemosynthesis, enabling the concise synthesis of complex steroidal products. Recent advancements in chemoenzymatic strategies, especially those involving P450 hydroxylase, 3-sterone-Δ¹-dehydrogenase, reductase, and enzyme cascades, have significantly contributed to the efficient and straightforward synthesis of complex steroid medications. On this basis, the future research opportunities and challenges are also discussed, aiming to provide a reference for the efficient development of more value-added steroid compounds, including the development of new generation steroid intermediates, the discovery of novel steroid biocatalysts, and the establishment of steroid synthesis pathways in mycobacteria.