Sex control techniques are in great demand for aquaculture because of the huge benefits of mono-sex production. In a previous study on yellow catfish (Tachysurus fulvidraco), intersex individuals were produced by administrating 17α-methyltestosterone (MT) or aromatase inhibitor letrozole (LZ). The ovarian and testicular portions of intersexual gonads developed well and were completely separated, arousing our interest in producing self-fertilizable fish and clone-like fish populations. In this study, yellow catfish fry were treated with a combination of MT and LZ at a low dose (5 mg kg−1 MT, 300 mg kg−1 LZ) at a low water temperature (27.0 °C, mixed sex-producing temperature, LL group) and a high temperature (33.5 °C, male-producing temperature, HL group), and with a high dose (10 mg kg−1 MT, 600 mg kg−1 LZ) at a low water temperature (LH group), during 12–65 days post-hatching, the critical period of sex differentiation. Both XX and XY intersex were produced with different percentages at the same time through co-treatment with MT and LZ, with or without the help of male-producing temperature. The highest percentage of XY intersex (53% in XY, 36% in XX and XY) and XX intersex (47% in XX, 25% in XX and XY) was produced in LH and HL groups, respectively. Results of gonadal morphology, histology, and sperm dynamic analysis indicate that some XX and XY intersex individuals were capable of self-fertilization through artificial insemination. The reproductive performance of these intersex individuals was then investigated by injecting spawning agents, dissecting gonads, and artificially fertilizing eggs with their own sperm. The XX, XY, and YY genotypic individuals were produced by the self-fertilization of XY intersex individuals. The present study successfully produced high percentages of XX and XY intersex and conducted self-fertilization by mating the eggs and sperm of a single intersex individual. Self-fertilization in fish will show great potential in aquaculture genetics and breeding, for example, generating highly homozygous (or ‘‘clonal’’) genetic material, producing mono-sex populations, and identifying unknown sex determination modes. In particular, the cloning method of producing genetic materials will be greatly beneficial for maintaining advantageous traits through self-fertilization generation after generation.