Currently, analyzing pharmaceuticals and biomarkers is crucial for ensuring medication safety and protecting life and health, and there is an urgent need to develop new and efficient analytical techniques in view of the limitations of traditional analytical methods. Molecularly imprinted ratiometric fluorescent (MI-RFL) sensors have received increasing attention in the field of analytical detection due to their high selectivity, sensitivity and anti-interference ability, short response time, and visualization. This review summarizes the recent advances of MI-RFL sensors in the field of pharmaceuticals and biomarkers detection. Firstly, the fluorescence sources and working mechanisms of MI-RFL sensors are briefly introduced. On this basis, new techniques and strategies for preparing molecularly imprinted polymers, such as dummy template imprinting, nanoimprinting, multi-template imprinting, and stimulus-responsive imprinting strategies, are presented. Then, dual- and triple-emission types of fluorescent sensors are introduced. Subsequently, specific applications of MI-RFL sensors in pharmaceutical analysis and biomarkers detection are highlighted. In addition, innovative applications of MI-RFL sensors in point-of-care testing are discussed in-depth. Finally, the challenges of MI-RFL sensors for analysis of pharmaceuticals and biomarkers are proposed, and the research outlook and development trends of MI-RFL sensors are prospected.

In this study, a RGB based ternary-emission molecularly imprinted ratiometric fluorescence (MI-RFL) sensor was facilely constructed by using a post-imprinting mixing strategy for the sensitive detection of enrofloxacin (ENR). Upon excitation at 365 nm, the MI-RFL sensor exhibited significant emission peaks at 450, 550, and 620 nm. As the ENR concentration increased, the blue fluorescence generated by ENR in the sensing system gradually intensified, while the red and green fluorescence emitted by the quantum dots in the molecularly imprinted polymers (MIPs) was significantly quenched. Sensing conditions were systematically investigated, including the excitation wavelength, mixing ratio of red/green MIPs, the pH of the buffer solution, and the reaction time. Under the optimal conditions, the developed sensor showed a good linear relationship within the range of 0.25–4 ppm along with obvious color change, with a low detection limit of 0.134 ppm. High selectivity was also attained with an imprinting factor up to 11.65. When applied to real samples of seawater and seafood, the sensor showed good recovery rates of 94.3–126.4% and accuracy with a relative standard deviation of less than 3.97%. Furthermore, the sensor-based kit was easily fabricated and, thus, naked-eye detection of ENR was realized onsite. This study can provide a universal approach for the rapid and visual detection of ENR in complicated matrices.