Spermatogenesis is the production of spermatozoa by the testes, a process that is associated with reproduction in males. Amphioctopus fangsiao is an economically important species from the northern coast of China, and little research has been done on spermatogenesis in this species. In this study, we performed transcriptome analysis of testis and other somatic tissues. The maturing stage of testis development was selected, in which the emergence of spermatozoa was observed. A total of 15,773 differentially expressed genes were identified, including 8,922 upregulated genes and 6,851 downregulated genes. GO and KEGG enrichment analyses were performed to explore the functions of genes related to spermatogenesis, and the interactions between genes were examined by constructing protein interaction networks. Pathways and genes related to spermatogenesis were identified, including the oxytocin signaling pathway, the calcium signaling pathway, the FOXO signaling pathway, the DNAH family, and other key genes. The expression levels of 25 key genes were verified by qRT-PCR. The results for spermatogenesis-related genes will provide insights into the processes of spermatogenesis and testis development in A. fangsiao.