The development of hyperpolarized bioresponsive probes for magnetic resonance imaging (MRI) applications is an emerging and rapidly growing topic in chemistry. A wide range of hyperpolarized molecular biosensors for functional MRI have been developed in recent years. These probes comprise many different types of small-molecule reporters that can be hyperpolarized using dissolution dynamic nuclear polarization and parahydrogen-induced polarization or xenon-chelated macromolecular conjugates hyperpolarized using spin-exchange optical pumping. In this Perspective, we discuss how the amplified magnetic resonance signals of these agents are responsive to biologically relevant stimuli such as target proteins, reactive oxygen species, pH or metal ions. We examine how functional MRI using these systems allows a great number of biological processes to be monitored rapidly. Consequently, hyperpolarized bioresponsive probes may play a critical role in functional molecular imaging for observing physiology and pathology in real time.