The biosynthesis of anthocyanins is influenced by external environmental conditions such as light, temperature, and nitrogen level, with nitrogen level being a key factor in anthocyanin synthesis and accumulation. Nitrogen level regulates the transcription factors involved in the anthocyanin synthesis pathway, with low nitrogen levels promoting anthocyanin accumulation, while high nitrogen levels have the opposite effect. Purple potatoes are a type of cultivated crop that is rich in anthocyanins and has unique economic value. Nitrogen fertilizer is crucial to improve the agronomic traits, yield, quality, and anthocyanin content of purple potatoes. In this study, the impact of four different nitrogen concentrations—0 kg/hm2 (N0), 90 kg/hm2 (N1), 225 kg/hm2 (N2) and 360 kg/hm2 (N3)—on the agronomic traits, yield, quality, and anthocyanin content of purple potatoes, ‘Huasong 66’, at different stages were investigated by using physiological index measurement and RNA-seq technology. It was found that the purple potato ‘Huasong 66’ was more sensitive to low nitrogen (N1). Under N1 level of nitrogen fertilization, ‘Huasong 66’ possessed the finest agronomic traits, yield, and quality, and the total anthocyanins in the tubers were significantly increased. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that nitrogen levels in purple potato tubers primarily affect genes related to nutrient transport and metabolism by regulating carbon and nitrogen metabolism, enzyme catalysis and binding, and signal transduction. In addition, nine candidate genes related to the anthocyanin synthesis pathway had been preliminarily screened. These results provide a basis to understand the impact of different nitrogen levels on the tuber yield and anthocyanin synthesis of purple potatoes.