Real-time polymerase chain reaction (real-time PCR) tests were successfully conducted in an aluminum-based microfluidic chip developed in this work. The reaction chamber was coated with silicone-modified epoxy resin to isolate the reaction system from metal surfaces, preventing the metal ions from interfering with the reaction process. The patterned aluminum substrate was bonded with a hydroxylated glass mask using silicone sealant at room temperature. The effect of thermal expansion was counteracted by the elasticity of cured silicone. With the heating process closely monitored, real-time PCR testing in reaction chambers proceeded smoothly, and the results show similar quantification cycle values to those of traditional test sets. Scanning electron microscope (SEM) and atomic force microscopy (AFM) images showed that the surface of the reaction chamber was smoothly coated, illustrating the promising coating and isolating properties. Energy-dispersive X-ray spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), and inductively coupled plasma-optical emission spectrometer (ICP-OES) showed that no metal ions escaped from the metal to the chip surface. Fourier-transform infrared spectroscopy (FTIR) was used to check the surface chemical state before and after tests, and the unchanged infrared absorption peaks indicated the unreacted, antifouling surface. The limit of detection (LOD) of at least two copies can be obtained in this chip.