Silver arowana, Osteoglossum bicirrhosum, is sexually monomorphic, and its sex determination mechanism is still poorly understood, posing a significant challenge to its captive breeding efforts. To this end, we assembled two high-quality chromosome-level genomes for both female and male silver arowana, with scaffold N50 values of 10.6 Mb and 10.4 Mb for female and male individuals, respectively. Combining re-sequencing data of 109 individuals, we identified two sex-specific SNPs and confirmed a ZW model of sex-determination in this ancient species. Both sex-specific SNPs are located in about 26-kb upstream of foxl2 gene. We subsequently propose a complex gene network in the sex determination process of silver arowana, with foxl2 acting as the central player. The sex chromosomes are homomorphic with a potentially recent origin, as linkage disequilibrium analysis showed minor recombination suppression. Furthermore, we successfully developed accurate molecular markers for sex identification of silver arowana at each developmental stage, significantly improving the reproductive efficiency and further promoting the industrial development.