l-Homocysteine has been an amino acid intermediate of interest for over 20 years due to its implication in various adverse health conditions, including cardiovascular disease. Here, we report the first in vitro selection and application of high affinity aptamers for the target l-homocysteine. Two novel aptamer sequences were selected following 8 rounds of selection that displayed high affinity binding and selectivity to homocysteine compared to other amino acids. One of the selected aptamers, Hcy 8 (KD = 600 ± 300 nM), was used to develop a gold-nanoparticle biosensor capable of sensitive and selective homocysteine detection in human serum, with a limit of detection of 0.5 μM and a linear range of 0.5-3.0 μM. This biosensor allows rapid detection of free homocysteine in human serum samples at low cost, with little preparation time and could be adapted to be part of a po
