The invention relates to a small perturbance stratified substrate sludge in-situ sampler used for collecting deposits in water and a sampling method thereof. The sampler consists of a C-shaped inner tube, a C-shaped outer tube, an outer tube conical head, an inner tube handle, an outer tube handle and an inflatable plug, wherein the outer tube and the inner tube are sleeved together and are respectively formed by fixedly connecting a plurality of sections; the inner tube can be rotated by the inner tube handle at the top so as to superpose or close C-shaped openings of the inner tube and the outer tube. The inner tube is firstly rotated so as to close the C-shaped opening when substrate sludge is fetched; the inner tube is rotated so as to open the C-shaped opening after being inserted by a predetermined depth, and then the inner tube and the outer tube are rotated after being inclined by a small angle together so as to rotate the substrate sludge into the inner tube. Then the inflatable plug is put on the sludge surface and is inflated; the inner tube is rotated to close the C-shaped opening; finally, the inner tube and the outer tube are lifted from the water and laid on the ground for stratified sampling. The invention has small perturbance to the substrate sludge, does not change stratified and physical properties of the substrate sludge, has light weight, portability, convenient operation and wide generalization and application values and , is suitable for sampling work of substrate sludge of hydraulic reclamation yards of river and lake.