For maintaining the healthy metabolic status, vitamin D is a beneficial metabolite stored majorly in its pre-activated form, 25-hydroxyvitamin D₃ (25(OH)D₃). Due to its important role in bone strengthening, the study was planned to quantify 25(OH)D₃ levels in our blood. Quantification techniques for 25(OH)D₃ are costly thus requiring a need for a low cost, and sensitive detection methods. In this work, an economic, and sensitive sensor for the detection of 25(OH)D₃ was developed using aptamer and graphene oxide (GO). Aptamer is an oligonucleotide, sensitive towards its target, whereas, GO with 2D nanosheets provides excellent quenching surface. Aptamer labeled with fluorescein (5’, 6-FAM) is adsorbed by π–π interaction on the GO sheets leading to quenching of the fluorescence due to Förster resonance energy transfer (FRET). However, in the presence of 25(OH)D₃, a major portion of aptamer fluorescence remains unaltered, due to its association with 25(OH)D₃. However, in the absence, aptamer fluorescence gets fully quenched. Fluorescence intensity quenching was monitored using fluorescence spectrophotometer and agarose gel based system. The limit of detection of 25(OH)D₃ by this method was found to be 0.15 µg/mL whereas when GO-COOH was used, limit of detection was improved to 0.075 µg/mL. Therefore, this method could come up as a new sensing method in the field of vitamin D detection.