Infectious diseases of the eye are one of the major causes of blindness. The rapid and accurate identification of pathogenic microorganisms plays an important role in clinical diagnosis and treatment. However, traditional methods for detecting pathogens have some disadvantages such as a long test time, low positive rate (or lack of specificity). As molecular biological detection technology with high sensitivity, especially the deoxyribonucleic acid amplification-based and sequencing-based detection methods have made great breakthroughs in detecting ocular pathogen microorganisms: specificity; speediness. The current state-of-the-art research and development in molecular detection technologies for ophthalmic pathogens was summarized, including polymerase chain reaction and its derivative techniques, gene chips, and metagenomic sequencing, and explored the application value and problems in the diagnosis and treatment of ocular surface infection disease and intraocular infection disease.