The coexistence of polystyrene nanoplastics (PS-NPs) and Cu²⁺ in wastewater can induce compound stress effects on microorganisms. This study investigated the effects of PS-NPs and Cu²⁺ on the growth, nitrogen removal, and biochemical characteristics of the aerobic denitrifying bacterium Pseudomonas stutzeri, and further elucidated the stress mechanism through transcriptomics analysis. The results showed that under exposure to 50 mg/L PS-NPs, bacteria growth activity increased by 25.3% compared to the control, accompanied by enhanced nitrogen removal and significant upregulation of genes related to ribosome function, the tricarboxylic acid (TCA) cycle, and ABC transport pathways. In contrast, under 10 mg/L Cu²⁺ stress, bacterial growth activity decreased by 83.2% compared to the control, with severe inhibition, cell membrane damage, and marked downregulation of ribosomal, nitrogen metabolism, and ABC transport pathways. Genes related to extracellular polymeric substances (EPS) secretion and cell membrane synthesis were significantly upregulated. Under combined stress, Cu²⁺ exerted the dominant inhibitory effect; however, PS-NPs promoted EPS secretion, which, along with PS-NPs themselves, adsorbed part of the Cu²⁺ ions and mitigated their toxicity.