Accurate quantification of low level of blood drugs by Latex enhanced immunoturbidimetric assay (LEITA) remains a challenge due to its inherent limited sensitivity. To deal with this problem, we designed a new agglutination-enhancement strategy, and applied it for development of a highly sensitive and accurate tacrolimus LEITA. By this principle, a very small amount of biotin labeled anti-tacrolimus monoclonal antibodies (BLATMA) can arise agglutination strong enough for accurate reading of the increased absorbance since the BLATMA bears multiple biotin molecules, and the agglutination mediated by BLATMA can be inhibited by a similarly small amount of tacrolimus when the drug binds to BLATMA, giving rise to an improved sensitivity. The limit of detection (LOD) and functional sensitivity obtained by the proposed tacrolimus LEITA was 0.22 ng/mL and 0.59 ng/mL, respectively, 8-20 times more sensitive than the conventional drug-latex or antibody-latex based direct inhibition LEITA formats. Good precision was observed in the whole range of clinically significant tacrolimus concentration. The reliability of the tacrolimus LEITA was demonstrated by its strong correlation with both liquid chromatography-tandem mass spectrometry (LC-MS/MS) (R2 = 0.977, slope = 0.998) and the ABBOTT tacrolimus chemiluminescent magnetic immunoassay (CMIA) (R2 = 0.982, slope = 1.01) in the analysis of 119 clinical samples. It's concluded that the agglutination-enhancement strategy can be applied to construct highly sensitive LEITA for accurate tacrolimus analysis; owing to the improved sensitivity, this technique can be expected not only to improve the reliability of LEITA for low-level drug monitoring, but also to broaden the scope of analytes detectable by LEITA.