On-site determination of trace copper ions in natural waters is of great significance to environmental monitoring, and how to develop accurate and specific point-of-care test methods is one critical issue. In the study, a paper-based analytical device (PAD) being modified with a new truncated DNAzyme (CLICK-T, which was derived from a reported DNAzyme-CLICK-17) was developed for Cu ions detection. The detection mechanism was based on Cu(II)-catalyzed azide-alkyne cycloaddition (Cu(II)AAC) reaction. It can directly conduct on-site analysis of Cu(II) ions based on fluorescent signals detected using a mobile phone. In the assay, the CLICK-T was firstly modified on the PADs. Then, water samples containing Cu ions mixed with 3-azido-7-hydroxycoumarin and 3-butyn-1-ol were instantly dripped on PADs and incubated for 20 min. Finally, the PADs were excited at 365 nm and emitted fluorescence which could be analyzed on site using smart phones. The Cu(II) concentration could be quantified through RGB analysis with the aid of iPhone APP software. The limit of detection is 0.1 µM by the naked eye due to the fact that CLICK-T exhibited a good catalytic effect on Cu(II)AAC. The Cu(II) concentration could also be directly detected without using reductant, such as ascorbic acid, which is prone to be oxidized in air. This simplifies the PDA detection process improves its efficiency. The PAD is convenient for the on-site analysis of Cu ions in natural waters.