Three enzyme-free electrochemical glucose (GLU) sensors with bimetallic structures were constructed by electrodeposition of gold and copper nanoparticles (AuNPs/CuNPs), nickel and copper nanoparticles (NiNPs/CuNPs), and a copper-based metal-organic framework (Cu-MOF) combined with PtNPs, respectively. Amongst, the Cu-MOF/PtNPs on a gold electrode (GE) exhibited the best catalytic performance for GLU oxidation due to the synergistic amplification. The relevant electrochemical behaviour of GLU showed that two OH- and two electrons were involved in the process. The differential pulse voltammetry (DPV) enables the establishment of linearity in the concentration of 0.40–25.00 mM, with a detection limit (LOD) of 0.06 mM and a sensitivity of 158.41 µA·mM−1cm−2. In addition, the Cu-MOF/PtNPs/GE sensor was used to detect GLU in oral liquid as well as human serum samples with a recovery rate of 97.00–103.98% and RSD less than 3.59%. Finally, a portable electrochemical detection system based on the STM32F103C8T6 microcontroller, obtained a GLU concentration 5.24 mM while detecting in a diluted injection where the value 5.40 mM was recorded by a commercial meter to show the system has very broad application prospects in the point-of-care setting.