l-proline (l-Pro) is the only imino acid among the 20 amino acids that constitute biological proteins, and its main hydroxylated product is trans-4-hydroxy-l-proline (T-4-Hyp). Both of them have unique biological activities and play important roles in biomedicine, food and beauty industry. With the in-depth exploration of the functions of l-Pro and T-4-Hyp, the demand for them is gradually increasing. Traditional methods of biological extraction and chemical synthesis are unable to meet the demand of “green, environmental protection and high efficiency”. In recent years, synthetic biology has developed rapidly. Through the intensive analysis of the synthetic pathways of l-Pro and T-4-Hyp, microbial cell factories were constructed for large-scale production, which opened a new chapter for the green and efficient production of l-Pro and T-4-Hyp. This paper reviews the application and production methods of l-Pro and T-4-Hyp, the metabolic pathways for microbial synthesis of l-Pro and T-4-Hyp, and the engineering strategies and advances on microbial production of l-Pro and T-4-Hyp, aiming to provide a theoretical basis for the “green bio-manufacturing” of l-Pro and T-4-Hyp and promote their industrial production.