A field experiment on “Response of mustard (Brassica juncea L.) to different bio-regulators under varying fertility levels” was carried out at Agronomy Instructional Farm, Chimanbhai Patel College of Agriculture, S. D. Agricultural University, Sardarkrushinagar during rabi 2017-18. With regard to the effect of different bio-regulators on mustard, all growth attributes studied during the course of investigation such as dry matter accumulation per plant, number of primary branches and secondary branches per plant showed significant improvement due to application of cycocel (CCC) @ 1500 ppm (B3). Whereas significantly the lowest plant height was exhibited under treatment B3. Similarly, treatments B3 (Cycocel @ 1500 ppm) and B1 (Brassinosteroid @ 0.5 ppm) remained comparable and exhibited significantly higher values of yield components and quality parameters viz., number of seeds per siliqua, number of siliquae per plant, test weight, seed yield per plant, protein content and oil yield. Application of cycocel @ 1500 ppm (B3) also significantly increased N and S content in seed and stover and also resulted in significant improvement in uptake of N, P, S and Zn in seed, stover and total uptake followed by treatment B1 (Brassinosteroid @ 0.5 ppm). However, harvest index, phosphorus and zinc content in seed and stover as well as available nutrients status in the soil (N, P2O5, S and Zn) did not influenced due to application of different bio-regulators. Treatment B3 (Cycocel @ 1500 ppm) realized the highest net return (₹ 64,090/ha) followed by treatment B1 (Brassinosteroid @ 0.5 ppm). The results of the present investigation indicated that all the growth attributes in terms of plant height, number of primary and secondary branches per plant; yield attributes viz., number of seeds per siliqua, number of siliquae per plant, test weight, seed yield per plant, seed, stover yield and quality parameters viz., oil content, oil yield and protein content showed significant improvement due to application of balanced fertilization treatment N4 (N50P50S40Zn1.7), though it was at par with treatment N3 (N50P50S40). Application of N50P50S40Zn1.7 (N4) registered significantly higher N, P2O5, S and Zn content (%) in seed and stover and seed, stover and total uptake by mustard followed by treatment N3 (N50P50S40). Similarly, application of N50P50S40Zn1.7 (N4) noted significantly higher available N (180.38 kg/ha), P2O5(39.68 kg/ha), S (8.49 mg/kg) and Zn (0.46 mg/kg), being at par with treatment N3 (N50P50S40) except available Zn status in soil. From economic point of view, the maximum net return (₹ 64,897/ha) and benefit cost ratio (3.13) were received with application of 50 kg N + 50 kg P2O5+ 40 kg S + 1.7 kg Zn/ha (N4) followed by treatment N3 (N50P50S40).