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Versatile sensing matrixes are essential for the development of enzyme-immobilized optical biosensors. A novel three-dimensional titanium dioxide nanotubes/alginate hydrogel scaffold is proposed for the detection of sweat biomarkers, lactate, and glucose in artificial sweat. Hydrothermally synthesized titanium dioxide nanotubes were introduced to the alginate polymeric matrix, followed by cross-linking nanocomposite with dicationic calcium ions to fabricate the scaffold platform. Rapid colorimetric detection (blue color optical signal) was carried out for both lactate and glucose biomarkers in artificial sweat at 4 and 6 min, respectively. The superhydrophilicity and the capillarity of the synthesized titanium dioxide nanotubes, when incorporated into the alginate matrix, facilitate the rapid transfer of the artificial sweat components throughout the sensor scaffold, decreasing the detection times. Moreover, the scaffold was integrated on a cellulose paper to demonstrate the adaptability of the material to other matrixes, obtaining fast and homogeneous colorimetric detection of lactate and glucose in the paper substrate when image analysis was performed. The properties of this new composite provide new avenues in the development of paper-based sensor devices. The biocompatibility, the efficient immobilization of biological enzymes/colorimetric assays, and the quick optical signal readout behavior of the titanium dioxide nanotubes/alginate hydrogel scaffolds provide a prospective opportunity for integration into wearable devices. The authors acknowledge support from Gobierno de Espana, Ministerio de Economia y Competitividad (Grant No. BIO2016-80417-P) (AEI/FEDER, UE), "Ministerio de Ciencia y Educacion de Espana" under grant PID2020-120313GB-I00/AIE/10.13039/501100011033 and the Basque Government (Grant IT1271-19). This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Sklodowska-Curie Grant Agreement No. 766007. The authors convey their special thanks to SGIker of the University of the Basque Country (UPV/EHU) and Dr. Javier G. Inanez for the technical equipment facility support. F.B.-L. and L.B.-D. acknowledge the "Red de Microfluidica Espanola" RED2018102829-T.