Abstract: Major invasive fungi include Aspergillus, Mucor, Candida, Cryptococcus and Pneumocystis, with Aspergillus and Mucor being the primary pathogens contributing to high mortality in IFD. Traditional diagnosic methods such as fungal culture and histopathological test are difficult to meet the requirement for early diagnosis due to their limitations like low sensitivity and long test time. There is an urgent clinical need for molecular diagnostic methods with high sensitivity and specificity to differentiate colonization from invasive infection, applicable to various samples (e.g., body fluids, tissues, blood). Additionally, ideal detective technologies should have the characteric of low cost, easy maintenance, and iterative updatability. As emerging molecular diagnostic technologies, polymerase chain reaction (PCR) and metagenomic next-generation sequencing (mNGS) demonstrate significant advantages in diagnosing invasive aspergillosis and mucormycosis due to their high sensitivity and specificity. This paper reviews the current applications, advantages, limitations and future directions of PCR and mNGS in diagnosing invasive aspergillosis and mucormycosis, providing insights into their potential.