Sensors that detect specific molecules of interest in a living organism can be useful tools for studying biological functions and diseases. Here, we provide a protocol for the construction of nanosensors that can noninvasively detect biologically important targets with magnetic resonance imaging (MRI). The key operating principle of these sensors is magnetic resonance tuning (MRET), a distance-dependent phenomenon occurring between a superparamagnetic quencher and a paramagnetic enhancer. The change in distance between the two magnetic components modulates the longitudinal (T1) relaxivity of the enhancer. In this MRET sensor, distance variation is achieved by interactive linkers that undergo binding, cleavage, or folding/unfolding upon their interaction with target molecules. By the modular incorporation of suitable linkers, the MRET sensor can be applied to a wide range of targets. We showcase three examples of MRET sensors for enzymes, nucleic acid sequences, and pH. This protocol comprises three stages: (i) chemical synthesis and surface modification of the quencher, (ii) conjugation with interactive linkers and enhancers, and (iii) MRI sensing of biological targets. The entire procedure takes up to 3 d.