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The reproductive process is usually controlled by the hypothalamic-pituitary-gonad (HPG) axis in vertebrates, while Kiss/GnRH system in the hypothalamus is required for mammalian reproduction but dispensable for fish reproduction. The regulation of FSH/LH expression in fish species is still unknown. Here, we identified miR-200 s on chromosome 23 (chr23-miR-200 s) as important regulators for female zebrafish reproduction. Knockout of chr23-miR-200 s (chr23-miR-200 s-KO) resulted in dysregulated expression of lhb and some hormone genes in the pituitary as revealed by comparative transcriptome profiling, leading to failure of oocyte maturation and ovulation as well as defects in reproductive duct development. Chr23-miR-200 s mainly expressed in the pituitary and regulated lhb expression by targeting the transcription repressor wt1a. Injection of hCG could rescue the defects of oocyte maturation in chr23-miR-200 s-KO zebrafish, whereas GnRH or LHRH-A2 could not, suggesting that Chr23-miR-200 s regulated lhb expression in a GnRH-independent pathway. It was remarkable that either injection of carp pituitary extraction (CPE), or co-injection of hCG with synthetic Oxytocin and Vasotocin could greatly rescue the defects of both oocyte maturation and ovulation in chr23-miR-200 s-KO zebrafish. Altogether, our findings highlight an important function of chr23-miR-200 s in controlling oocyte maturation by regulation LH expression, and Oxytocin and Vasotocin are potentially responsible for the ovulation in fish species.