The present invention provides a two-step Gram staining method. The method is conducted by using a clean slide, followed by adding 10˜30 μl primary stain from a reagent kit, adding an equivalent amount of specimens uniformly on said slide, then immediately adding 30˜60 μl counter-stain on said slide for incubating 15 seconds, rinsing with water, and finally examining under a microscope, the result appears color contrast on the center of the slide between the Gram-negative bacteria performing light red color and the Gram-positive bacteria performing deep purple color.