The presence of paraquat in the environment poses a danger to human health, leading to a growing demand for an uncomplicated and highly responsive method to detect paraquat. This work reports a new, simple, and sensitive colorimetric aptasensor based on the designed aptamers containing 1–5 paraquat binding sites (R1–R5) in combination with gold nanoparticles (AuNPs). Although the aptamers with more binding sites exhibited greater paraquat interaction capability, the aptasensor based on the R3 aptamer showed the highest detection sensitivity for paraquat in a linear range of 5–50 nM with a limit of detection of 1.29 nM, meaning that it is 2.14 fold more sensitive than the R1-aptasensor. This R3-aptasensor selectively detected paraquat but not the other tested herbicides, including difenzoquat, 2,4-D, ametryn, atrazine, and glufosinate. Also, it efficiently detected paraquat spiked in water samples within the precision acceptance criterion of recovery rates (96.8–105.0%) and the relative standard deviations (1.50–3.81%). These results demonstrated the development of a new aptasensor for paraquat detection, in which the multiple paraquat binding sites of the aptamers could enhance detection sensitivity.