The invention provides a method for preparing a three-dimensional paper chip based on a multi-edition patterning and silk screen combined printing technology. The method comprises the following specific preparation procedures: printing a hydrophobic printing solution on paper through a patterned ink-printing silk screen by a scraper so as to allow the hydrophobic printing solution to permeate through the paper and form a hydrophobic area, and taking an unprinted part as a hydrophilic area, so as to obtain a channel layer and a detection layer of the paper chip; printing a pretreating reagent on pretreatment areas of the channel layer and the detection layer through a patterned reagent-printing silk screen by a scraper; printing a detection reagent on a detection area of the detection layer through the patterned reagent-printing silk screen by a scraper; printing a glue solution on the channel layer through a patterned glue-printing silk screen by a scraper; and binding and assembling the channel layer and the detection layer, thereby obtaining the three-dimensional paper chip. According to the method provided by the invention, the process is simple, patterning is easy to realize, the accuracy of pattern making is high, instruments and equipment are simple, industrialization is easy to achieve, and the production cost is reduced remarkably.