Molecularly imprinted polymers (MIPs) in the micron and submicron scale based on the bifunctional cross-linker N,O-bismethacryloylethanolamine (NOBE) have been synthesized using bulk and miniemulsion polymerization, respectively. MIPs with distinct selectivity for the template testosterone were obtained. Colloidal MIP particles made using the miniemulsion technique have significant advantages compared to bulk MIP counterparts owing to their small size, homogeneity, and increased surface, as is demonstrated by optical batch rebinding studies using a nonimprinted polymer (NIP) as a negative control. Affinity and selectivity studies were also performed with the miniemulsion colloidal MIPs. These MIPs display largely increased imprint factors (6.8 vs 2.2) when compared to their bulk MIP counterparts. Further, selectivity studies by using analogue steroids show that colloidal MIPs also display a higher selectivity. In summary, miniemulsion MIPs show much better performance with respect to molecular recognition in aqueous solution, while providing at the same time the possibility for a water-based MIP synthesis.