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Screen-printing technology is a game changer in many fields including electrochemical biosensing. Two-dimensional nanomaterial MXene Ti₃C₂T_x was integrated as a nanoplatform to immobilise enzyme sarcosine oxidase (SOx) onto the interface of screen-printed carbon electrodes (SPCEs). A miniaturised, portable, and cost-effective nanobiosensor was constructed using chitosan as a biocompatible glue for the ultrasensitive detection of prostate cancer biomarker sarcosine. The fabricated device was characterised with energy-dispersive X-ray spectroscopy (EDX), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). Sarcosine was detected indirectly via the amperometric detection of H₂O₂ formed during enzymatic reaction. The nanobiosensor could detect sarcosine down to 7.0 nM with a maximal peak current output at 4.10 ± 0.35 × 10⁻⁵ A using only 100 µL of a sample per measurement. The assay run in 100 μL of an electrolyte showed the first linear calibration curve in a concentration window of up to 5 μM with a slope of 2.86 μA·μM⁻¹, and the second linear calibration curve in the range of 5–50 μM with a slope of 0.32 ± 0.01 μA·μM⁻¹ (R² = 0.992). The device provided a high recovery index of 92.5% when measuring an analyte spiked into artificial urine, and could be used for detection of sarcosine in urine for at least a period of 5 weeks after the preparation.