Background: Lipases are serine hydrolases that degrade triglycerides, an attribute that treasures wide applications in biodiesel production, detergent, chemical industries, etc. The most sought after the application is in the high quality and economical production of biodiesel under mild reaction conditions and simplified product separation. For the said application, fungal lipases are ideal catalysts that could effectively catalyze esterification and transesterification reactions with their specific ability to release fatty acids from 1, 3 positions of acylglycerols. Results: In the present work, to facilitate bulk synthesis, lipase production using Aspergillus niger MTCC 872 was studied by solid-state fermentation (SSF). The chosen fungal strain was evaluated for lipase production using a mixture of agroindustrial substrates viz. rice husk, cottonseed cake, and red gram husk in various combinations at flask level. Tri-substrate mixture (rice husk, cottonseed cake, and red gram husk) combined in the ratio of 2:1:1 has shown the maximum lipase activity 28.19 U/gds at optimum cultivation conditions of temperature 40°C, moisture content 75% (v/w), pH 6.0 and initial spore concentration of 5.4 million spores per mL. Further studies were performed for scale-up of lipase from flask level to lab scale using tray fermenter. Lipase activity was found to be 24.38 U/gds and 21.62 U/gds for 100 g and 1000 g substrate respectively. Conclusion: This is the first report on the production of lipase from Aspergillus niger MTCC 872 using tri-substrate mixture of rice husk (RH), cottonseed cake (CSC), and red gram husk (RGH). Moreover, comparison between individual, binary, and tri-substrate mixture was carried out for which the highest lipase activity was observed for tri-substrate mixture. In addition, comparable results were found when scale-up was performed using tray fermenter. Thus, the current work signifies usage of agro-industrial residues as substrates for enzyme production by solid-state fermentation process as an effective alternative to submerged fermentation for industrial applications.