Vancomycin quantification in milk and serum-like media requires more than target recognition because proteins, salts, lipids, carbohydrates, and nonspecific surface transport can shift the same aptamer-gating response in different directions. This study asks whether a printed carbon–gold impedimetric aptasensor can assign matrix-corrected vancomycin concentrations by coupling percentage charge-transfer-resistance opening with recovery factors measured in each sample class. The analytical record contained electrode geometry, gold-nanostructure deposition conditions, aptamer-affinity measurements, blocked and target-exposed resistance states, calibration constants, recoveries in three milk-fat levels and artificial serum, and interferent-retention responses. The printed strip had a geometric working-electrode area of 0.196 cm2 and an electroactive area of 0.226 cm2. Gold nanostructures were electrodeposited from 10 mM tetrachloroauric acid in acidic histidine solution at −100 μA for 600 s, followed by immobilization of a thiolated vancomycin aptamer and blocking with 6-mercapto-1-hexanol. Percentage opening of the receptor layer was converted to concentration using a linear relation from 0.05 μM to 1.00 μM, with a slope of 56.83% per micromolar, an intercept of -0.853%, R2 = 0.981, a detection limit of 1.721 nM, and a quantification limit of 5.216 nM. At a 1.00 μM spike level, recoveries were 99.10% in 0.1% fat milk, 98.10% in 1.5% fat milk, 104.40% in 3.0% fat milk, and 96.20% in artificial human serum. Matrix correction returned the concentration estimates to 0.996 μM–0.999 μM. Lactose, glucose, and gentamicin produced only 9.78–13.03% independent response, while vancomycin mixtures retained 89.05–96.58% of the target signal. The results show that this aptasensor supports concentration assignment only when the impedance opening, calibration interval, recovery factor, and selectivity retention are evaluated together for the stated matrix.