In this study primary culture, subculture₁ and subculture₂ of ovarian tissue from goldfish was done to develop an in vitro system of ovarian cells in this species. A goldfish weighing 42g and 7 cm in length was anesthetized by Clove powder (0.2 g/lit) and its ovary was removed and put in a petri dish containing 5ml L-15 medium, Streptomycin sulphate (200 ug/ml), Penicillin G potassium (Gibco, 200 iu/ml) and Amphotericin B (5 ug/ml). The oocytes which were in advanced maturation stage (IV) were isolated from ovary and the ovary cut into small pieces (explants) by a 5ml syringe. The ovary explants cultivated in 5ml L-15 medium supplemented with 15% FBS, Streptomycin sulphate (100 ug/ml), Penicillin G potassium (Gibco, 100 iu/ml) and Amphotericin B (2.5 ug/ml) at  25^°C. After two weeks when confluent monolayer formed on the bottom of the flask, the cells were washed with PBS containing antibiotics, dislodged by Trypsin-EDTA solution (0.25%) and cultured in 5ml of L-15 medium, FBS (15%), antibiotics at 25 °C (first subculture). After confluent monolayer formation, on the sixteenth day similar procedure was followed for the second subculture.The emerging cells from explants and two subcultures exhibited fibroblast-like cells.