β-Hydroxy-α-amino acids (HAAs) are a class of important chiral intermediates and have a wide range of uses in the pharmaceutical industry. Due to its adjacent chiral centers, it has attracted much attention to exploring strictly stereoselective biosynthesis methods. Threonine aldolase (TA) can catalyze the aldol reaction in one step for the synthesis of HAAs under mild conditions. TAs also exhibit a broad substrate spectrum and can catalyze the condensation of various aldehydes and amino acids, thereby constructing rich HAA libraries with immense potential for industrial applications. In this review, we have summarized the research progress of TA, including new enzyme mining, structure, catalytic mechanism analysis, high-throughput screening methods, protein engineering, and synthesis applications. Notably, we mainly focus on the research achievements of TA in structure-function relationship, mechanism analysis, and protein engineering. Currently, the catalytic process of TA has been elucidated, where it catalyzes the aldol reaction through the Schiff base exchange mechanism. Additionally, researchers have proposed diastereoselectivity regulating mechanisms of TA such as the “pathway hypothesis” and “dual conformation hypothesis”, which provide a foundation for unraveling the mystery of TA diastereoselectivity regulation. Moreover, significant progress has been made in TA molecular evolution, with multiple mutants obtained that showed strict diastereoselectivity synthesis for various chiral HAAs and their intermediates. Furthermore, we have discussed the current challenges and prospects of TA, which will guide for accelerating the industrial application of TAs as tool enzymes for synthesizing high-value HAAs compounds.