Vanilla is the main natural flavouring agent used in industries such as pharmaceuticals, food, flavouring, and fragrance, in which vanillin is the major component. Vanillin (4-hydroxy-3-methoxybenzaldehyde) is a secondary metabolite of plants and the major organoleptic aroma component of natural vanilla. The vanillin compound can be produced using the following routes: direct vanilla bean extraction, chemical synthesis, and biotechnological processes (bio-vanilla production). Nowadays, the chemical synthesis method used for vanillin production has been rejected by the United States and European legislation, while plant-derived vanillin is expensive. The current study demonstrates vanillin production from pumpkin peels (Cucurbita moschata) by Aspergillus niger via one-step fermentation approach. This study implements different concentrations of sodium hydroxide (1.0 M and 2.0 M) during alkaline hydrolysis pretreatment and different feeding volumes of hydrolysates during the biotransformation processes of ferulic acid into vanillin, classified as small feeding volumes (SFV) and large feeding volumes (LFV). Detection and quantification analysis were carried out using high performance liquid chromatography (HPLC), resulting in vanillin yield of 0.49 mg/L (1.0 M SFV), 0.5 mg/L (1.0 M LFV), 0.33 mg/L (2.0 M SFV), 0.59 mg/L (2.0 M LFV). Analysis with ultraviolet-visible (UV-VIS) spectrophotometry using thiobarbituric acid as reagent was carried out as well, resulting in vanillin yield of 2.76 µg/ml (1.0 M SFV), 3.78 µg/ml (1.0 M LFV), 2.68 µg/ml (2.0 M SFV), 3.05 µg/ml (2.0 M LFV). In conclusion, pumpkin peels can be considered a great source of ferulic acid and Aspergillus niger was reported as an efficient fungus in converting ferulic acid to vanillic acid, which will then be transformed into vanillin.