By employing fluorescein and AuNPs as energy donors and acceptors, respectively, a novel fluorescence resonance energy transfer (FRET)-based dual-mode sensor for selective recognition and quantitative detection of thiourea was designed and constructed in this study for the first time. Herein, it is demonstrated that fluorescein could be adsorbed on the surface of AuNPs and induce fluorescence quenching through the well-known FRET process. The preferential introduction of thiourea would reduce the overall surface negative charges of AuNPs by replacing the original citrate groups, leading to the aggregation of AuNPs. Meanwhile, thiourea could prevent the binding between fluorescein and AuNPs, reduce the as-formed FRET effect, and then lead to fluorescence recovery of the fluorescein. Therefore, a dual-mode sensor with AuNP-related colorimetric and fluorescein-based fluorescence readout was rationally developed. Under the optimum conditions, the detection limits were calculated to be 10 nM and 23 nM for fluorescent and colorimetric sensors, individually, and a limit of 0.4 μM was detected by the naked eye. Finally, such a simple, convenient, cost-effective, highly selective and sensitive sensing assay was successfully applied in the detection of thiourea in tap water and fruit juice samples.