The invention discloses a urine multi-index detection microfluidic device. The device includes the following parts: an inlet channel, which is located in the upstream of the device; two or more flow channels, which are communicated with the inlet channel and located in the downstream of the inlet channel; a reaction zone, which is located in the flow channel, and includes analysis test paper; a liquid collection chamber, which is in the downstream of the reaction zone and communicated with the downstream end of the flow channel. An upper passageway formed by the inlet channel, the flow channel, the reaction zone and the liquid collection chamber comprises a first passageway layer and a middle layer. A plurality of layers can operate to detect multiple indexes; and the processes of urine taking, reaction, detection and analysis can be completed in a short time, so as to greatly simplify the operation steps, save time and increase the efficiency. In addition, the invention also discloses a preparation method of the device. The method adopts a piece of three-dimensional drawing software to design a passageway model and combines with the three-dimensional printing passageway model, so as to prepare the device in a rapid and cost-saving way.