Rapid and accurate detection of changes in glucose (Glu) and hydrogen peroxide (H2O2) concentrations is essential for the predictive diagnosis of diseases. Electrochemical biosensors exhibiting high sensitivity, reliable selectivity, and rapid response provide an advantageous and promising solution. A porous two-dimensional conductive metal–organic framework (cMOF), Ni-HHTP (HHTP = 2,3,6,7,10,11-hexahydroxytriphenylene), was prepared by using a one-pot method. Subsequently, it was employed to construct enzyme-free paper-based electrochemical sensors by applying mass-producing screen-printing and inkjet-printing techniques. These sensors effectively determined Glu and H2O2concentrations, achieving low limits of detection of 1.30 μM and 2.13 μM, and high sensitivities of 5573.21 μA μM−1cm−2and 179.85 μA μM−1cm−2, respectively. More importantly, the Ni-HHTP-based electrochemical sensors showed an ability to analyze real biological samples by successfully distinguishing human serum from artificial sweat samples. This work provides a new perspective for the use of cMOFs in the field of enzyme-free electrochemical sensing, highlighting their potential for future applications in the design and development of new multifunctional and high-performance flexible electronic sensors.