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Here, we show the utility of the fluorescent biosensor <i>h</i>CaM-M124C-<i>mBBr</i> in detecting and determining the affinity of serotonin (5-HT). We obtained a <i>K</i>_d of 5-HT (0.71 μm) for the first time, the same order of magnitude as most anti-CaM drugs. This data can contribute to understanding the direct and indirect modulation of CaM on its binding proteins when the 5-HT concentration varies in different tissues or explain some of the side effects of anti-CaM drugs. On the other hand, molecular modeling tools help the rational design of biosensors and adequately complement the experimental results. For example, the docking study indicates that 5-HT binds at the same site as chlorpromazine (site 1) with a theoretical <i>K</i>_i of 2.84 μM; while the molecular dynamics simulations indicate a stability of the CaM–5-HT complex with a theoretical Δ<i>G</i> of −4.85 kcal mol⁻¹, where the enthalpy contribution is greater. Thus, the combination of biotechnology and bioinformatics helps in the design and construction of more robust biosensors.