Paper-based ratiometric fluorescence sensors are normally prepared using two or more types of fluorescent materials on a paper chip for simple, low-cost and fast detection. However, the choice of multi-step and one-step modifications on the paper chip affects the analytical performance. Herein, a novel paper-based dual-emission ratiometric fluorescence sensor was designed for the selective detection of tetracycline (TC). Carbon dots (CDs) modified with Eu3+ were combined with a sealed paper-based microfluidic chip by two methods: one-step grafting of CDs-Eu3+ on paper and step-by-step grafting of CDs and Eu3+ on paper. The analytical performance was studied and optimized respectively. The red fluorescence of Eu3+ at 450 nm is enhanced and the blue fluorescence of CDs at 617 nm is quenched by energy transfer in the presence of TC. Under optimal conditions, TC is selectively determined in the linear range from 0.1 μM to 100 μM with a detection limit of 0.03 μM by the step-by-step grafting method. In addition, the sealed paper chip could effectively prevent pollution and volatilization from the reagent. This technique has been used to analyze TC in seafood aquaculture water with satisfactory results.