The invention discloses a method for preparing a brain tissue frozen section, belonging to the fields of biology and medical experiments. The method comprises (1) a step of tissue extraction, immobilization and dehydration, namely a step of subjecting an animal brain tissue to immobilization and gradient dehydration; (2) a step of freezing pretreatment, namely a step of washing the brain tissue with a PBS solution, wiping the brain tissue, leveling the brain tissue, wrapping the brain tissue with tin foil paper, freezing the brain tissue in a rapid freezing zone of a freezing microtome, and then taking the tissue out for subsequent treatment; (3) a step of semi-embedding treatment, namely a step of subjecting the brain tissue to semi-embedded treatment, and allowing the tissue to stand for1 minute; (4) a step of frozen sectioning, namely a step of fixing a sample holder to a probe of the microtome, and performing continuous coronal frozen sectioning; and (5) a step of section collection, namely a step of collecting sections of desired positions into a six-pore plate containing a frozen preserving fluid, and storing the sections at -20 DEG C for long time. Semi-embedding treatmentis adopted to treat the brain tissue, and therefore, influences of an embedding agent on the brain tissue are avoided, and sections are complete and free of ice crystals, and have good immunofluorescent staining properties.