The main issues of imaging ellipsometry-based biosensing for small molecules are the low sensitivity and narrow detection range due to the low molecular weight of small molecules that results in a negligible signal. To meet this challenge, we theoretically investigated the deciding factors of the ellipsometry signal and further applied the theory to guide the design of ellipsometry-based biosensor using metal nanoparticles that have a high dielectric constant. Significant signal amplification effects can be achieved by using nanoparticle labels including magnetic nanoparticles and gold nanoparticles. Guided by the theory, we have developed a sensitive surface-enhanced imaging ellipsometry (SEIE)-biosensor for detecting chloramphenicol in real milk sample with high sensitivity (with a limit of detection of 6 pg/mL) and broaden detection range. This nanoparticles-enabled SEIE not only greatly improves the sensitivity of conventional imaging ellipsometry-based biosensors but also retains the advantages of conventional methods in terms of automated and convenient operation, providing an effective strategy for detection of trace small molecules in complex samples that holds great promise in scientific research, clinical diagnosis, and food safety.