Thiamine detection has been developed using Copper-Metal Organic Frameworks (CuMOF) and Rhodamine B-complexed Graphene Oxide (RhGO), facilitating both direct and indirect fluorescence-based sensing of thiamine. Both these methods are less cumbersome and sensitive fluorometric methods which are developed from less expensive materials. CuMOF offered the oxidation route of thiamine to thiochrome, which is a fluorescence compound; through which thiamine was detected conveniently by measuring thiochrome. On the other hand, rhodamine B bound graphene oxide–RhGO, released rhodamine B upon interaction with the thiamine through the competitive binding against the rhodamine B located on the graphene oxide, which allowed us to develop a facile sensor for thiamine in DMF. Moreover, the sensitivity of the sensor was improved through the encapsulation effect with cucurbit[7]uril (CB[7]). The limits of detection (LOD) for CuMOF and RhGO with CB[7] were determined to be 48.39 × 10–8 M and 68.33 × 10–8 M, respectively. The sensing ability of RhGO was effectively utilized in commercially available thiamine drugs, and its performance was evaluated against other hydrochloride drugs such as metformin, ciprofloxacin, and cetirizine demonstrating its suitability for real-time sample analysis. This approach provides a practical solution for both analytical and pharmaceutical laboratories. Overall, two different detection methods for thiamine have been developed with good selectivity and sensitivity.