Recently, luminescent metal-organic framework (MOF) materials have attracted considerable attention in fluorescence sensing. In this essay, we prepared a new cluster-based CaII-MOFs {[Ca1.5(μ8-HL1)(DMF)2]·DMF}n (1) with good water dispersibility, excellent photoluminescence properties (FL quantum yield of 20.37%) and great fluorescence stability. Further, it was employed to design as an "off-on" fluorescence sensor for sensitive detection of l-cysteine. This proposed strategy was that fluorescence of CaII-MOFs 1 was quenched for providing a low fluorescence background by the introduction of Pb2+ forming the CaII-MOFs 1/Pb2+ hybrid system. The quenching effect could be ascribed to the static quenching mechanism because of the formation of ground-state complexes and coordination interactions between the free carboxyl of H4L1 ligands of CaII-MOFs 1 and Pb2+. Then, with the addition of l-cysteine into the CaII-MOFs 1/Pb2+ hybrid system, the fluorescence signal was immediately restored. This result was because the Pb2+ was gradually released from the hybrid system by chelation interactions between the -SH groups of l-cysteine and Pb2+. This method received a relative wide linear range varying from 0.05 to 40 μM and a low detection limit of 15 nM for detection of l-cysteine. This proposed strategy was also successfully applied to detect l-cysteine in human serum samples with satisfactory recoveries from 95.9 to 101.5%.