Dopamine (DA) plays a significant role in the human body and cerebral nervous system, and the accurate and rapid assay of DA is essential for the diagnosis of related diseases. Herein, we proposed a turn-on ratiometric fluorescent DA assay strategy by integrating a specific DA–resorcinol chemical reaction with a multifunctional lanthanide metal–organic framework (Ln-MOF). First, Eu–BTC (1,3,5-benzenetricarboxylic acid) was synthesized and further modified to obtain Cu@Eu–BTC, which simultaneously plays multiple roles such as fluorescence internal standard, nanoreactor, cooperative catalysis effect and color shift enhancement. The Cu@Eu–BTC dispersion-based method exhibits ultra-sensitive (limit of detection, LOD is 0.01 μM) and wide-range linear response (0.04–30 μM) to DA in real serum. More importantly, it has excellent selectivity for DA, even in the presence of epinephrine and norepinephrine analogs. Thus, this method realizes the accurate and precise quantification of DA in serum (recoveries: 98.1%–110.1%, relative standard deviation RSD < 4.6%). Next, Cu@Eu–BTC was prepared into paper microchip, which has good storage stability (RSD < 3.5%, n = 3) in four weeks and achieves point-of-care visual DA assay coupled with smartphone-assisted portable detection device. The MOF paper microchip-based method shows low sample consumption (30 μL), high accuracy and precision for the quantification of DA in serum (recovery of 92.9%–106.2%, RSD < 5.3%), and gets the same assay results as the MOF dispersion-based method (relative error ≤ 6.83%). To our knowledge, this is the first time to propose the catalytic fluorescence turn-on detection strategy of DA based on a MOF paper microchip.