MicroRNAs (miRNAs) are endogenous 22 nucleotide small non coding RNAs in eukaryotes genomes, which play a very important role in gene expression regulation. Sexual dimorphism miRNA screening would help in understanding gonad development mechanism. In this study, we firstly isolated small RNAs from amphioxus Branchiostoma japonicum gonad. By microarray analysis, we totally identified 36 miRNAs, which were abundantly expressed in amphioxus gonad. Among them, 11 miRNAs were regarded as sexual dimorphism miRNAs in amphioxus. Six miRNAs (bfl-miR-92b, bfl-miR-10A06, bfl-miR-281, bfl-miR-36B03, bfl-miR-29B02 and bfl-miR-26A01）specific to amphioxus were regarded as sexual dimorphism miRNAs, among which, 4 miRNAs showed high expressions in testis whereas 2 miRNAs exhibited high expressions in ovary tissue. In addition, bfi-miR-92b, bfl-miR-10A06, bfl-miR-281 and bfl-miR-36B03 were identified as high abundantly expressed miRNA in amphioxus testis by using RT qPCR techniques. All these results provided a basis for sexual dimorphism miRNA function identifications in the future.