Aurantiochytrium not only excels in producing long-chain polyunsaturated fatty acids such as docosahexaenoic acid for humans, but it is also a source of essential fatty acids with minimal impacts on wild fisheries and is vital in the transfer of atmospheric carbon to oceanic carbon sinks and cycles. This study aims to unveil the systems biology of lipid production in the Australian Aurantiochytrium sp. TC20 by comparing the transcriptomic profiles under optimal growth conditions with increased fatty acid production from the early (Day 1) to late exponential growth phase (Day 3). Particular attention was paid to 227 manually annotated genes involved in lipid metabolism, such as FAS (fatty acid synthetase) and subunits of polyunsaturated fatty acids (PUFA) synthase. PCA analysis showed that differentially expressed genes, related to lipid metabolism, efficiently discriminated Day 3 samples from Day 1, highlighting the key robustness of the developed lipid-biosynthesis signature. Highly significant (pFDR < 0.01) upregulation of polyunsaturated fatty acid synthase subunit B (PFAB) involved in fatty acid synthesis, lipid droplet protein (TLDP) involved in TAG-synthesis, and phosphoglycerate mutase (PGAM-2) involved in glycolysis and gluconeogenesis were observed. KEGG enrichment analysis highlighted significant enrichment of the biosynthesis of unsaturated fatty acids (pFDR < 0.01) and carbon metabolism pathways (pFDR < 0.01). This study provides a comprehensive overview of the transcriptional landscape of Australian Aurantiochytrium sp. TC20 in the process of fatty acid production.