Due to the relatively high lipid productivity, microalgae are promising raw material for biofuel production. The understanding for the mechanism of lipid biosynthesis is an important concern which may contribute to the increase of lipid production. Chlamydomonas reinhardtii under nitrogen depletion condition is a model system to investigate the pathway of lipid biosynthesis. Substantial amount of data have been accumulated using “omics” approaches recently, however, the identification of reference and biomarker proteins in nitrogen depletion-mediated triacylglycerol biosynthesis in C. reinhardtii is limited. In this study, C. reinhardtii CC-124 grown in control and nitrogen depleted medium were surveyed to compare the morphology, cell density, lipid content, and total protein content at multiple time points (0, 1, 2, 4 and 6 days). Under nitrogen depletion treatment, the culture turned yellow from green and the A₇₅₀ and cell number was decreased, indicated that the cell growth was retarted. Furthermore, the concentration of neutral lipid was up-regulated based on nile red staining assay, while the coomassie brilliant blue staining assay indicated that the concentration of total protein was decreased. Western blot (WB) was performed to detect the expression patterns for 20 candidate C. reinhardtii proteins at different time points. To identify the reference proteins under nitrogen depleted condition, the Pearson’s correlation coefficient (PCC) between the content of total proteins and candidate proteins was calculated. Three proteins (Histone H3, RBCL (ribulose-1, 5-bisphosphate carboxylase/oxygenase large subunit) and BCR1 (biotin carboxylase, ACCase complex 1)) were selected due to their significant positive correlations (P < 0.01) in both nitrogen depleted and control conditions. Comparison of average relative fold change (ARF) of the candidate proteins indicated that the top 3 proteins are ATPs-β (ATP synthase CF1 beta subunit), GAP2 (glyceraldehyde 3-phosphate dehydroase 2) and RMT1 (rubisco large subunit N-methyltransferase 1), their ARFs are 180.59, 52.90 and 12.48, respectively, which were then chosen as biomarkers. Futhermore, to compare the earliest time point at which biomarker band can be detectable, protein samples at early stage (0, 2, 4, 8, 12, 18, 24 and 48 h) at nitrogen depletion treatment were collected and analysed by WB, it was showed that the induction bands of ATPs-β, GAP2 and RMT1 appeared at 8, 18 and 12 h, respectively. Thus, ATPs-β was the most appreciate biomarker since it is the earliest showed and the largest degree varied among candidates proteins tested. The reference and biomarker proteins identified in this study will provide help for the mechanism investigation of nitrogen depletion responses and lipid biosynthesis in C. reinhardtii, the expression profiling acculumated for candidate proteins can be refered by the reseach community.