Dopamine (DA) and Fe as the important bioactive ingredients, playing an indispensable role in human metabolism. Therefore, developing the accurate detection of DA and Fe is of great significance for disease screening. Herein, we put forward a simple, rapid, and sensitive fluorescent detection strategy for the detection of dopamine and Fe based on Rhodamine B-modified MOF-808 (RhB@MOF-808). RhB@MOF-808 produced strong fluorescence at 580 nm, and the fluorescence was significantly quenched after DA or Fe was added, which was regarded as a static quenching process. Detection limits are as low as 60.25 nM and 48.34 nM, respectively. Furthermore, based on the responses of DA and Fe to the probe, molecular logic gates were successfully designed. More importantly, RhB@MOF-808 had excellent cell membrane permeability and had been successfully used to label DA and Fe in Hela cells, which presented a potential biological application value as a fluorescent probe for detecting DA and Fe.