Using stenospermocarpic grapes as female parents could improve the seedless ratio of offspring via embryo rescue techniques, so they are widely used for breeding seedless grapes. Here, we report the variation in phenotype, endogenous hormones, and internal cellular structure of ovules during embryo abortion in ‘Thompson Seedless’. Moreover, we used the stenospermocarpic grapes as female parents to breed new seedless grapes. Our result indicated that the seed coat of abnormal ovules turned brown from 38 to 42 days after flowering (DAF). In hormones level, two ratios of endogenous hormones’ content [gibberellic acid (GA₃) + indole-3-acetic acid (IAA)]/abscisic acid (ABA) and zeatin riboside (ZR) /abscisic acid (ABA) had decreased by 36 DAF. In paraffin sections of embryo sac, indicated that there were few or no embryos in ovules were observed from 37 to 42 DAF. Importantly, the globular embryos in ‘Thompson Seedless’ were significantly smaller than those in ‘Ruby Seedless’ at 40 DAF, indicating the former had a lower embryo rescue efficiency as a female parent; accordingly, the most suitable time for embryo cultured in vitro was 39 DAF for ‘Thompson Seedless’. Result from embryo rescue, demonstrated that 5 mg/L exogenous brassinolide (BR) promoted embryo recovery, embryo germination, and polyembryony formation of ‘Flame Seedless’. Concurrently, WPM medium containing 1 µM of 6-BA helped embryos germinate into normal plantlets and reduced the abnormal plantlet development percentage. A total of 3573 hybrid ovules and 402 new progeny plants were obtained using the stenospermocarpic grapes as female parents crossed with disease-resistant Chinese wild grapes, among which 212 progeny with seedless trait and 23 with disease-resistant were detected by molecular markers, respectively.