We present a novel reflectance-based colorimetric aptasensor using gold nanoparticles for the detection of oxytetracycline for the first time. It was found that the reflectance-based measurement at two wavelengths (650 and 520 nm) can generate more stable and sensitive signals than absorbance-based sensors to determine the aggregation of AuNPs, even at high AuNP concentrations. One of the most common antibacterial agents, oxytetracycline (OTC), was detected at concentrations as low as 1 nM in both buffer solution and tap water, which was 25-fold more sensitive, compared to the previous absorbance-based colorimetric aptasensors. This reflectance-based colorimetric aptasensor using gold nanoparticles is considered to be a better platform for portable sensing of small molecules using aptamers.