Disclosed are an ammonia-oxidizing bacterium screening and testing reagent box, a screening and testing method and a bacterium sample separating method. The ammonia-oxidizing bacterium screening and testing reagent box comprises a bacterium strain enrichment tube, ammonia-oxidizing testing tubes 1, 2 and 3, bacterium strain preservative liquid and color developers A and B. The screening and testing method includes inoculating bacterium strains to be detected to the ammonia-oxidizing testing tubes 1, 2 and 3 to culture after enriching the bacterium strains to be detected via the bacterium strain enrichment tube, and storing enrichment bacterium liquid by the bacterium strain preservative liquid simultaneously; adding the color developer A and B after culturing of the ammonia-oxidizing testing tubes and judging ammonia-oxidizing capability of the bacterium strains according to color development states. The bacterium sample separation method includes culturing bacterium samples, diluting, applying and separating. The ammonia-oxidizing bacterium screening and testing reagent box, the screening and testing method and the bacterium sample separating method has the advantages that effect of quickly screening and testing ammonia-oxidizing bacteria in high throughput is realized, one-stop operations of culturing, testing and preserving of the bacterium strains are realized.