Polystyrene nanoplastics (PS-NPs) and Cu2+ coexistence in wastewater can induce a compound stress effect. The growth, nitrogen removal, and biochemical characteristics of aerobic denitrifying bacteria (Pseudomonas stutzeri) were investigated under PS-NPs and Cu2+ stress conditions, while the stress mechanism was elucidated through transcriptomics analysis. The results showed that under the stress of 50 mg/L PS-NPs, the bacteria exhibited an 25.3% increase in growth activity compared to the control group, accompanied by enhanced nitrogen removal activity and significant upregulation of ribosome, TCA cycle, and ABC transport pathway. Under the stress of 10 mg/L Cu2+, the growth activity of the bacteria decreased by 83.2% compared to the control, and the bacteria was significantly inhibited. The cell membrane was damaged, and the metabolic pathways related to ribosome, nitrogen metabolism, and ABC transport were significantly downregulated. The genes related to extracellular polymeric substances (EPS) secretion, cell membrane synthesis were significantly upregulated. Under compound stress conditions, Cu2+ stress exerted dominant effects. However, PS-NPs exhibited the ability to stimulate enhanced EPS production. Additionally, both PS-NPs and EPS demonstrated the capacity to adsorb a portion of Cu2+, thereby alleviating the detrimental effects caused by Cu2+ on the bacteria. These results provide theoretical insights into utilizing aerobic denitrifying bacteria for treating nanoplastics and heavy metal wastewater.