Immune checkpoint inhibition is an important strategy in cancer therapy.Blockade of CTLA-4 and PD-1/PD-L1 is well developed in clinical practice.In the last few years,LAG-3 has received much interest as an emerging novel target in immunotherapy.It was recently reported that FGL1 is a major ligand of LAG-3,which is normally secreted by the liver but is upregulated in several human cancers.FGL1 is a crucial biomarker and target for cancer immunotherapy.As the efficacy of immunotherapy is limited to specific types of patients,the subset of patients needs to be selected appropriately to receive precise treatment according to different biomarkers.To date,there is no test to accurately assess FGL1 expression levels.Nanobodies have some outstanding features,such as high stability,solubility and affinity for diagnostic and therapeutic applications.Here,we report the development and validation of a rapid,sensitive,and cost-effective nanobody-based immunoassay for the detection of FGL1 in human serum.In this study,human FGL1 recombinant protein was expressed and purified for the first time as an immunized antigen.Then,we constructed a nanobody phage display library and screened several nanobodies that bind FGL1 with high affinity.We selected two nanobodies targeting different epitopes of FGL1,one as a capture and the other conjugated with HRP as a probe.The double nanobody-based sandwich ELISA to detect the concentration of FGL1 showed a good response relationship in the range of 15.625-2000 ng/mL,and the recoveries from the spiked sample were in the range of 78％ and 100％.This assay could be used as a potential approach for evaluating FGL1 expression for patient stratification and for predicting the therapeutic efficacy of targeting the LAG3/FGL1 axis.