Biological aging is an independent risk factor for many cardiovascular diseases; some are treated with β-blockers that may protect dysfunctional endothelium during aging by increasing NO, decreasing ONOO-, and restoring NO/ONOO- balance. A nanotechnological approach was used to simultaneously monitor NO and ONOO- produced by a single aortic endothelial cell from Wistar-Kyoto rats of different ages. β-blockers (metoprolol and atenolol) were administered 2 weeks before the animals were sacrificed. Nanosensors were placed near the endothelium, and calcium ionophore- stimulated NO and ONOO- release was measured. Endothelial nitric oxide synthase (eNOS) undergoes uncoupling with aging, manifested by a decrease in NO (from 503 ± 12 to 163 ± 5 nmol/L) and a 3-fold increase in ONOO- for 16-week-old and 110- week- old rats, respectively. Metoprolol reversed eNOS uncoupling, increased the production rate and concentration of NO, and increased an overall ratio of NO]/ONOO-]. This effect was not observed with atenolol, but L-arginine, sepiapterin, and superoxide dismutase were beneficial.