In this work, a simple and label-free fluorescent method via fluorescent indicator displacement (FID) was proposed for enantioselectively determining d-enantiomer of arginine vasopressin (DV) using DV-specific DNA aptamer (V-apt) and one guanidiniophthalocyanine dye (Zn-DIGP). Zn-DIGP that preferentially binds to single-stranded DNA with fluorescence enhancement rather than duplexes occupies the long internal loop of V-apt and generates intensive fluorescence. Then DV is introduced into the solution containing Zn-DIGP and V-apt, and displaces the Zn-DIGP from the binding site of internal loop, leading to fluorescence decrease. But l-enantiomer cannot induce any fluorescence change due to the selectivity of V-apt. This established FID technique can detect DV with a detection limit of 100 nM and exhibits a broad linear range, and is able to discriminate enantiomers of arginine vasopressin unambiguously. Moreover chiral separation by chromatography, complicated experimental procedures and covalent modification of tags (such as organic dyes, redox-active metal complexes) are avoided in our strategy. This simple and label-free method is promising for fabricating diverse aptasensors to determine other biomolecules and drugs.