Antibiotic determination at low concentrations is a critical challenge to ensure water, food, and environmental safety. This work reports the application of molecularly imprinted nanoparticles (MINs) as synthetic antibodies in a microplate assay for tetracycline (TC) determination at subnanomolar levels. MINs with hydrodynamic radii ranging from 200 to 300 nm were synthesized via solid-phase imprinting on magnetic nanoparticles using lymecycline (LMC) as an auxiliary template. MINs immobilized onto polystyrene microplates exhibited a selective binding of the conjugate between LMC and horseradish peroxidase (HRP), yielding analytical responses up to fourfold higher than those observed in blank wells. MINs saturated with LMC-HPR showed the quantitative displacement of the conjugate upon incubation with LMC or TC, yielding concentration-proportional responses from 0.001 to 100 nmol L-1. For LMC, we obtained limits of detection (LOD) between 0.018 and 0.020 nmol L-1 and limits of quantification (LOQ) from 0.054 to 0.061 nmol L-1. For TC, the LOD values ranged between 0.001 and 0.006 nmol L-1, with LOQ values of 0.004-0.019 nmol L-1. Within the studied concentration range, the analytical responses of MINs were two to five times higher than those obtained from nonimprinted materials or blank controls. These findings support the performance of MINs as antibody mimetics with high recognition capacity, relatively low production costs, and robust analytical applications.