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Nowadays, there is great interest in the use of microfluidic paper-based analytical devices (μPADs) for low-cost diagnostics. In most cases, the test equipment is needed. Here we report a new type of device-independent detection method based on timer-paper-based analytical devices, which can be tested by smartphone, and its application for cholesterol detection. The Quick Response code was designed as the timer component of this device. Wax printing method was employed to print the pattern on filter paper. The color and enzyme reagents have been immobilized in the hydrophilic channel to complete the colorimetric detection for cholesterol. Under laminar flow conditions of the cellulose network, the liquid volume of detection zone has been quantified by monitoring the fluid residence time on different area of the timer-paper-based devices. The precise monitoring of detection time can promote the accuracy of colorimetric detection. This is very important for the quantitative detection of paper-based analytical devices. One significant outcome of this report is that simple and accurate timer can be used for detection process self-clocking. The factors of total detection time have been investigated. The linear range of the calibration curve was 3.0 ~ 6.0 mmol L−1, with correlation coefficient of 0.9956. With the characteristic of easy to use, low cost and accurate monitoring of detection time, this kind of timer-paper-based devices can be applied to cholesterol or other substances rapid detection.