The invention discloses a gradient centrifugation separation method of total microbes in porcine gastrointestinal contents and manure. The gradient centrifugation separation method comprises the following steps: A, performing reagent preparation of a separation buffer solution; B, performing an experimental method as follows: (1) selecting the porcine gastrointestinal contents and manure and aseptically collecting contents and manure in stomach, jejunum, ileum, cecum and colon; (2) respectively weighing the weight of the contents and manure, placing into a sterile centrifugal tube, centrifuging and discarding supernatant; (3) adding the separation buffer solution into the centrifugal tube, shaking up on a vortex mixer and centrifuging; (4) taking out the centrifugal tube, pouring supernatant fluid into the centrifugal tube, centrifuging the centrifugal tube loaded with the supernatant fluid and discarding the supernatant; (5) weighing the separation buffer solution, adding into a centrifugal tube a, shaking up on the vortex mixer, centrifuging and discarding the supernatant; (6) respectively repeating the step 3, the step 4 and the step 5 twice; (7) pouring away the supernatant fluid, transferring thalli into a 2ml centrifugal tube and preserving at the temperature of -70 DEG C for determining the dry weight of the thalli and a microbial flora. The method is easy, simple and convenient to operate and the cost is low.