Abstract: The utilization value of female haploids derived from unpollinated ovaries is superior to male haploids derived from anthers or microspores in tobacco breeding. Therefore, in vitro culture of unpollinated ovary is of great significance for tobacco haploid breeding. In order to establish the technique system for in vitro culture of unpollinated ovaries, different F1 hybrids were used as test materials to explore the effects of six key factors, including disinfection method, inoculation method, inoculation density, inorganic salt concentration, exogenous hormone ratio, and agar concentration, on embryoid induction in HW media. The results showed that the measures that contributed to the induction of embryoids were as follows:the buds were immersed in 0.1% HG solution for 8 min without peeling the petals, the ovary walls were peeled off and the ovaries were transected before inoculation, 2 ovaries were inoculated in each 125 mL triangular flask, and the inorganic salt concentration in HW media was increased by an additional 60%, with additional IAA 0.5 mg/L and 6-BA 2 mg/L, or KT 2.0 mg/L and 2, 4-D 0.5 mg/L, and agar concentration of 9 g/L. Based on this, the technical parameters for in vitro culture of unpollinated tobacco ovaries were further improved.