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In this paper, diamond film-coated silicon was developed to be used as a substrate in a microstrip gas chamber (MSGC) for the first time. The roughness of the composite substrate was rather low, and the resistivity was in the range of 1010–1011Ω cm. Its capacitance was very small and almost had no variation with frequency. All these results prove that the diamond film/Si composite material is a promising substrate for MSGCs. Using this composite substrate, a MSGC detector with an area of 2 × 2 cm2 was fabricated. The effects of the drift voltage and cathode voltage on the energy resolution for 5.9 keV 55Fe x-rays have been examined in detail and discussed. An energy resolution (the relative full width at half maximum of the x-ray peak) of 12.3% was achieved when the MSGC was operated at a drift voltage of −1000 V and a cathode voltage of −650 V with a gas mixture (90% Ar + 10% CH4).