BACKGROUNDIn response to growing concerns regarding heavy metal contamination in food, particularly chromium (Cr)(VI) contamination, this study presented a simple, sensitive and practical method for Cr(VI) detection.RESULTSA magnetic separation‐based capture–exponential enrichment ligand system evolution (SELEX) method was used to identify and characterize DNA aptamers with a high affinity for Cr(VI). An aptamer, Cr‐15, with a dissociation constant (Kd) of 4.42 ± 0.44 μmol L−1 was obtained after only eight rounds of selection. Further innovative methods combining molecular docking, dynamic simulation and thermodynamic analysis revealed that CrO42− could bind to the 19th and 20th guanine bases of Cr‐15 via hydrogen bonds. Crucially, a label‐free fluorometric aptasensor based on SYBR Green I was successfully constructed to detect CrO42−, achieving a linear detection range of 60–300 nmol L−1 with a lower limit of detection of 44.31 nmol L−1. Additionally, this aptasensor was able to quantitatively detect CrO42− in grapes and broccoli within 40 min, with spike recovery rates ranging from 89.22% to 108.05%. The designed fluorometric aptasensor exhibited high selectivity and could detect CrO42− in real samples without sample processing or target pre‐enrichment.CONCLUSIONThe aptasensor demonstrated its potential as a reliable tool for monitoring Cr(VI) contamination in fruit and vegetable products. © 2024 Society of Chemical Industry.