Seahorse is a kind of precious marine medicinal teleosts with special father’s rearing pattern and gonadal structure. Analyzing the histological structure of seahorse gonads is significant of improvement in efficiency of artificial breeding. Compared with paraffin section, frozen section has a fast and simple operation, while the quality of which is susceptible to multiple factors. The fixative is a key factor for Hematoxylin-Eosin (HE) staining. In our study, we chose 8 kinds of fixatives for the frozen section of seahorse ovary and compared the quality of HE stain, results showed that glacial acetic acid fixation was an ideal fixative for frozen sections of seahorse ovary stained with HE, which displayed a clear tissue structure with distinct nuclear boundary, high nucleo-plasmic contrast and bright-coloured yolk granules.