This article describes, for the first time, a highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for therapeutic monitoring and pharmacokinetic studies of lenalidomide (LND), the potent drug for treatment of multiple myeloma (MM). The assay employed a polyclonal antibody that specifically recognizes LND with high affinity, and LND conjugate of bovine serum albumin (LND-BSA) immobilized onto microplate wells as a solid phase. The assay involved a competitive binding reaction between LND, in plasma sample, and the immobilized LND-BSA for the binding sites on a limited amount of the anti-LND antibody. The assay limit of detection was 0.05 ng/mL and the effective working range at relative standard deviations (RSD) of ≤5% was 0.1-20 ng/mL. Analytical recovery of LND from spiked plasma was 100.98 ± 3.05. The precisions of the assay were satisfactory; RSD was 2.96-6.67% and 4.46-7.14%, for the intra- and inter-assay precision, respectively. The procedure is convenient, and one can analyze ∼200 samples per working day, facilitating the processing of large-number batch of samples in clinical laboratories. The proposed ELISA has a great value in routine analysis of LND for its therapeutic monitoring and pharmacokinetic studies.