At present, based on whole genome sequencing, sequences and genes annotation of the sheep (Ovis aries) Y chromosome are still absent. The isolation of Y chromosomes followed by sequencing has been approved as an effective approach to analyze this complex chromosome in other species. In this study, we established a highly efficient synchronization method for G2/M phase of sheep fibroblasts, which was successfully applied to flow-sorting chromosomes of sheep, with a focus on isolation and sequencing of the ovine Y chromosome. The isolated (~80,000) Y chromosomes were verified by fluorescence quantitative real-time polymerase chain reaction, further confirmed by fluorescence in situ hybridization, and amplified by the MALBAC method before next-generation sequencing. The sequence results indicated that 68.90% of reads were Y chromosome-related sequences as they are homologous to the bovine Y chromosome. The remaining 31.1% of reads were aligned to the sheep reference genome, including 13.57% reads to chromosome X and 6.68% to chromosome 17. Importantly, the paired-end reads that are properly aligned to the bovine Y sequence assembly accounted for 46.49%, indicating the success in the ovine Y chromosome isolation and the high quality of the Y chromosome sequences. This study not only set up a foundation for future sequencing, assembly and annotation of the ovine Y chromosome, but also provide a validated approach to overcoming difficulties in sequencing Y chromosome in other mammalian species.