Dry eye (DE) is a highly prevalent ocular surface disease which affects the quality of life and results in low working efficiency. Frequent instillation is required due to low bioavailability of conventional eye drops. The aim of this study is to develop a novel formulation of tacrolimus (TAC), routinely prescribed for DE, by combination of the microcrystal technology and layer-by-layer assembly. First, nonspherical tacrolimus microcrystals (TAC MCs) are synthesized by antisolvent-induced precipitation. These TAC MCs are modified by alternate deposition of poly(allylamine hydrochloride) (PAH) and carboxymethyl cellulose (CMC) subsequently to obtain CMC-coated TAC MCs (TAC-(PAH/CMC) ). The resultant formulations are evaluated in vivo in a mouse DE model induced by an intelligently controlled environmental system. Compared with commercially available TAC eye drops and the TAC MCs counterpart, TAC-(PAH/CMC) exhibits superior therapeutic performance with reduced drug instillation frequency, which is attributed to the nonspherical geometry of MCs, the lubricant, mucoadhesive effect of CMC, and the anti-inflammatory function of TAC. Therefore, TAC-(PAH/CMC) represents a better option for the management of DE.© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.