Under nitrogen stress conditions, Nannochloropsis spp. accumulate large amounts of lipids in the form of triacylglyceride (TAG). Although the mechanism of this process has been confirmed from the perspective of transcriptomics and metabolomics, post-translational modification (PTM) analysis is still limited, hampering the understanding of the dynamic adaptation to nitrogen deficiency. In this study, phosphoproteomics data for 3 h, 6 h, 12 h, 24 h, 48 h, and 10 d under nitrogen-depleted (N−) and nitrogen-replete (N+) conditions were obtained and integrated with previous proteomics and transcriptomics data for Nannochloropsis oceanica. The result revealed that 1371 phosphorylation sites were identified. The top three functions of the whole changed phosphorites were gene expression (167 sites), protein synthesis (134 sites), and transport (112 sites). Meanwhile, a total 126 phosphorylated proteins were identified as members of metabolic pathways with biological activities as diverse as lipid metabolism, carbon fixation, photosynthesis, and the tricarboxylic acid (TCA) cycle. Furthermore, 11 proteins and 6 phosphorylated proteins were detected in the process of TOR signalling pathway; it provides evidence that phosphorylation events play an important role in mediating the response to nitrogen starvation through modulation of the TOR signalling cascade. Temporal cluster analysis unveiled that phosphorylation modification plays dual regulatory roles in modulating the cell’s adaptive response to early nitrogen starvation, namely the coordination between phosphorylation-mediated signalling and metabolic regulation. Overall, the integration of phosphorylation PTM analysis with multi-omics data provides novel insights into how Nannochloropsis adapts to nutrient stress with relevance for future biotechnological applications.