Quantifying creatinine (Cn) in biological fluids is crucial for clinically assessing renal insufficiency, thyroid irregularities, and muscle damage. Therefore, it is crucial for human health to have a simple, quick, and accurate Cn analysis technique. In this study, we have successfully synthesized a 3D ratiometric dual-metal−organic framework, namely, the amino-MIL-53@Mo/ ZIF-8 and rhodamie B heterostructure, using an internal strategy for sustained growth. The dual-MOF functions as an adsorbent and preconcentrates Cn. The pH, reaction time, and volume ratio of amino-MIL-53@Mo/ZIF-8/rhodamie B were optimized using the one-variable-at-a-time technique in this study. The quantitative study of the Cn concentration for this RF biosensor was obtained under ideal conditions (R 2 = 0.9962, n = 3), encompassing the linear range of 0.35−11.1 μM. The detection and quantitation limits were 0.18 and 0.54 nM, respectively. Both intra-and interday reproducibility showed high repeatability of the RF biosensor, UV−vis, and ZETA potential studies, and the Stern−Volmer relationship was used to clarify the fluorescence quenching process. These superior sensing capabilities and the benefits of simple manufacturing, acceptable stability, and practicality make the RF biosensor intriguing for ultrasensitive Cn detection in practical applications.