Abstract: In recent years, real-time fluorescence quantitative polymerase chain reaction (qPCR) technology has become an essential tool for molecular diagnosis, pathogen detection, and gene expression analysis, thanks to its high sensitivity, speed, and real-time quantification capabilities. In 2022, the global market size of nucleic acid testing-related products and services, including instruments, reagents, consumables, and after-sales service support, reached 7.3 billion US dollars, with PCR-based technologies accounting for 66.7% of the market share and exhibiting a consistent growth trend. Although qPCR technology has been widely applied across multiple fields, the preclinical development of diagnostic kits—a process that includes primer design and reaction system optimization—still faces such issues as unclear procedures, non-standardized methods, and inconsistent evaluation criteria. Herein, we reviewed the guidelines, key resources, and standardized processes of qPCR assay reagent development, aiming to provide theoretical support for improving the efficiency and quality control of assay reagent development, and to discuss future directions for the optimizing and improving qPCR technology in the context of artificial intelligence.