The commercial production of microalgae-derived docosahexaenoic acid (DHA) has been expanding due to several advantages of algal over fish oil produced DHA. While the DHA production technology from microalgae is already economically competitive, alternatives to pure glucose and yeast extract still need to be explored to reduce the costs and increase the profits further. In the present study, orange peel waste (OPW) and various nitrogen sources were investigated as alternative nutrient sources for the economic cultivation of Aurantiochytrium sp. KRS101. The utilization of orange peel extract (OPE) supplemented with NaNO3 showed a higher DHA yield than that supplemented with NH4Cl or urea, and pH 5.5 was found to be the optimum initial condition for Aurantiochytrium sp. KRS101. OPE optimized with supplemental NaNO3 (1.2 g/L) resulted in a DHA yield of 0.63 g/L, which was 2.5 fold greater than the yield obtained using a conventional basal medium containing a similar amount of total nitrogen and 67% greater total carbon sources. This result implied not only that the conventional use of glucose and yeast extract have lower efficiency levels during nutrient metabolism but also that the types of carbon and nutrient sources have a significant effect on the DHA yield. The addition of supplemental glucose further enhanced the biomass, fatty acid methyl esters (FAME), and DHA yields, which unveiled the high C:N ratio requirement of Aurantiochytrium sp. KRS101. These results suggest that the development and optimization of microalgae fermentation using OPE and NaNO3 is a possible route for the economical production of DHA and for the additional utilization of food waste.