Phosphinothricin (PPT) is one of the top three herbicides, known for its broad spectrum, high herbicidal activity, and non-selectivity, with a highly optimistic market prospect. However, PPT exists in two enantiomers (D-PPT and L-PPT), with the herbicidal activity primarily stemming from L-PPT. Therefore, efficient synthesis of L-PPT with high optical purity is crucial. Pesticide manufacturing enterprises have attempted to develop chemical synthesis methods for L-PPT using approaches such as racemic compound splitting, asymmetric synthesis, natural amino acid chiral source method, and chiral auxiliary induction. However, due to challenges such as low stereo-selectivity, low product yield, and high production costs, large-scale production has not been achieved. Under the guidance of Academician Yin-Chu Shen, the “Father of Biopesticides in China”, our research group has conducted scientific research and industrial practice on the biosynthesis of L-PPT for over 20 years. In cooperation with multiple enterprises, we have developed more than ten process routes and technologies. Among them, five routes (racemic mixture derivatization-resolution route, racemic PPT-chiral separation route, generic compound cyanation followed by hydrolysis route, de novo synthesis from common chemicals route, and synthesis of homoserine followed by chemical synthesis) are discussed in detail in this review. Each route’s reconstruction, establishment of bioinorganic amine technology, creation of biocatalysts, high-density fermentation for enzyme production, product separation and purification, and reaction equipment are included. Notably, we developed the BioHPP^®, a biomanufacturing technology for the synthesis of highly optically pure L-PPT. Based on this technology, a ten-thousand-ton digital and intelligent production line for L-PPT was established. Utilizing smart sensors and actuators, real-time data collection, transmission, analysis, and feedback adjustment were achieved at over a thousand control points. This led to fully automated parameter collection and control, increasing production efficiency by 50% and reducing labor intensity by more than 70%, thereby realizing the bio-intelligent manufacturing of ten thousand tons of L-PPT. Based on the long-term accumulation of our research efforts, we summarize and analyze the mainstream production processes of D,L-PPT, detailing on the principles and methods of biomanufacturing technology and synthetic biology to construct the key synthesis system for L-PPT. We also compare the characteristics and key points of industrialization implementation of these routes in terms of substrate synthesis and selection, types of biocatalysts, use of amino donors, and separation and purification. It can be foreseeable that, with the aid of synthetic biology technology, an increasing number of high-optical-purity chiral pesticides will be produced on a large scale through biomanufacturing in the future.