A microfluidic channel structure and a fabrication method thereof, a microfluidic detecting device and a detecting method thereof are disclosed. The microfluidic channel structure includes a support portion; a foundation portion, provided on the support portion and including a first foundation and a second foundation spaced apart from each other; and a channel defining portion, provided on a side of the foundation portion that is away from the support portion and including a first channel layer and a second channel layer, the first channel layer covering the first foundation and the second channel layer covering the second foundation have a gap therebtween to define a microfluidic channel; and the first channel layer and the second channel layer are made of a same material.