Lanthanide-based luminescent materials have gained huge attention due to their applications in optoelectronic devices, sensing, bio-imaging, anti-counterfeiting, and more. In this work, we report a luminescence-based sensor for the detection of tryptophan using orotic acid-capped Tb3+-doped CaSO4 nanorods (NRs). Orotic acid (OA) was found to play a dual role as a capping agent to control the growth of the nanorods and as a sensitizer for Tb3+ ions. The resulting nanorods exhibited excellent dispersibility and strong photoluminescence signals characteristic of Tb3+ ions in the visible region. Nearly 10-fold enhancement in the emission intensity was noted through OA sensitization compared to direct excitation of Tb3+ ions (acceptors). Interestingly, the strong emission intensity of the NRs reduced significantly with the addition of tryptophan. In contrast, hardly any change was noted with the addition of other amino acids and metal ions, suggesting greater selectivity for tryptophan. Moreover, there is barely any notable interference from other amino acids toward the detection of tryptophan. The limit of detection is found to be ∼0.61 μM. Finally, the sensing study was extended to biological samples to detect tryptophan present in blood plasma, urine, and saliva samples. The nanorods demonstrated high detection abilities, indicating the potential of the developed materials for biomedical applications.