We investigated the optimum co-culture ratio with the highest biological nitrogen removal rate, revealing that chemical oxygen demand, total nitrogen (TN), and ammoniacal nitrogen (NH3-N) removal was increased in theChlorella pyrenoidosaandYarrowia lipolyticaco-culture system at a 3:1 ratio. Compared with the control, TN and NH3-N content in the co-incubated system was decreased within 2–6 days. We investigated mRNA/microRNA (miRNA) expression in theC. pyrenoidosaandY. lipolyticaco-culture after 3 and 5 days, identifying 9885 and 3976 differentially expressed genes (DEGs), respectively. Sixty-five DEGs were associated withY. lipolyticanitrogen, amino acid, photosynthetic, and carbon metabolism after 3 days. Eleven differentially expressed miRNAs were discovered after 3 days, of which two were differentially expressed and their target mRNA expressions negatively correlated with each other. One of these miRNAs regulates gene expression of cysteine dioxygenase, hypothetical protein, and histone-lysine N-methyltransferase SETD1, thereby reducing amino acid metabolic capacity; the other miRNA may promote upregulation of genes encoding the ATP-binding cassette, subfamily C (CFTR/MRP), member 10 (ABCC10), thereby promoting nitrogen and carbon transport inC. pyrenoidosa. These miRNAs may further contribute to the activation of target mRNAs. miRNA/mRNA expression profiles confirmed the synergistic effects of a co-culture system on pollutant disposal.