Crayfishes (Cherax quadricairnatus) were collected from artificial ponds in Zhejiang Institute of Freshwater Fisheries. The samples were observed under microscope after being sacrificed and investigated with histological, histochemical, biochemical methods and chromatography. The C. quadricarinatus spermatophore comprises a highly convoluted tube and a firm membrane forming a cordlike mass, and this spermatophoric ribbon is embedded in a gelatinous matrix. The structure of C. quadricarinatus spermatophore was observed under the electron microscopy, which consists of sperm mass, spermatophore matrix and spermatophore wall. Sperm mass is embedded in a dense fibrillar matrix and with an acellular wall surrounding both of them. In this study, the structure of the spermatophore wall in the vas deferens was also investigated using electron microscopy and biochemical test. The wall of the spermatophore is composed of two concentric layers: a thin primary spermatophore layer which is a fibrillar structure that directly surrounds the sperm mass and spermatophore matrix, and a thick secondary layer which contains heterogeneous vesicles and loose filament surrounds the primary spermatophore layer. The vas deferens of C. quadricarinatus consists of an anterior vas deferens, a middle vas deferens, and a posterior vas deferens. The histology of the inner epithelial cells of the vas deferens contributing to the spermatophore mass is described. The inner epithelial cell of the coiling anterior vas deferens secretes the weakly acidophilic filament that forms the equable primary layer of spermatophore. Neutral muopolysaccharides and protein are the main components of the primary layer. There are incrassate epithelial cells of middle vas deferens, which are fasciculate high cellula columnoepithelialis. In the middle of epithelial cells, some secretory vacuoles were observed with acidophily vesicular secretion, which formed the inequable secondary layer. Acidic muopolysaccharides and protein are the main components of the secondary layer. Spermatophores structure are complete in posterior vas deferens which has a simpler structure than the former two but incrassate muscular layer and larger tube. The posterior vas deferens links with ejaculatory duct. Between the posterior vas deferens and the ejaculatory duct, there is a bottleneck with two valves inside. The ejaculatory duct has an incrassate muscular layer and protuberant inner epithelial cells. No spermatophore in the ejaculatory duct is visible.