The invention discloses a device and method for enrichment and separation of nitrifying bacteria. The device includes a nitrification treatment tank, a partition plate is arranged in the nitrification treatment tank and divides the nitrification treatment tank into an enrichment tank and a separation tank. An aeration device is arranged at the bottom of the enrichment tank, a membrane module is arranged above the aeration device, the enrichment tank is internally filled with two fillers, the specific gravity of the first filler is 0.95-0.98 g/L, the particle size is 3-5 cm, the specific gravity of the second filler is 1-2.2 g/L, and the particle size is 0.15-0.3 mm. A filter screen for separating the first filler and the second filler is arranged at the lower portion of the separation tank, a first filler collecting port and a water draining port are formed above the filter screen, and a second filler collecting port is formed below the filter screen. A water draining sluice is arranged on the partition plate and located above the filter screen. The device can simultaneously achieve enrichment and separation of nitrifying bacteria and is high in nitrifying bacterium enrichment purity and large in enrichment amount.