Nanobody，derived from the variable domain of heavy chain only antibodies（VHHs），is the smallest antigen recognition unit．It is an ideal candidate for the next generation of immunoassays due to its high structural stability and high expression yields compared with the conventional antibody．This study aimed to investigate effects of the multivalency and gene fusion of anti-aflatoxin B,1,（anti-AFB,1,） nanobody on the bioactivities of recombinant proteins．The anti-AFB,1, nanobody，namely G8，was screened from an immune VHH phage display library in a previous work．The coding sequence of G8 was cloned into the vector pET30 between the restriction enzyme sites ,Nde,Ⅰ and ,Sfi,Ⅰ，generating vector pET30－G8．The overlap PCR was performed to assemble the tandem structures（DiG8 and TriG8） with flexible linker（GGSGG）．The overlap PCR products encoding DiG8 and TriG8 were purified，and cloned into the vector pET30 at the same site as the G8，generating vector pET30－DiG8 and pET30－TriG8，respectively．To construct expression vectors for the recombinant fusion proteins，the green fluorescent protein （GFP） encoded gene was firstly amplified by PCR with specific primers F0 and R0，and then subcloned into the restriction sites between ,Sfi ,Ⅰ and ,Not ,Ⅰ of vectors pET30－G8，pET30－DiG8，and pET30－TriG8，respectively. The resulting vectors（pET30－G8－GFP，pET30－DiG8－GFP，and pET30－TriG8－GFP），which fused GFPs at the C terminals of monomeric，dimer and trimer anti-AFB,1, nanobody，were confirmed via colony PCR，restrictive digestion，and sequencing．The vectors were transformed to ,Escherichia coli, BL21 （DE3），respectively．To induce the expression of the target proteins，isopropyl ,β,-D-1-thiogalactopyranoside（IPTG） was added to the culture．Fusion proteins each contain six histidine tag at their C-terminals which were purified by affinity chromatography，respectively．SDS－PAGE analysis showed that the three fusion proteins（G8－GFP，DiG8－GFP and TriG8－GFP） were solubly expressed in ,E. coli ,yielding at least 6.0，7.5，4.0 mg/L，respectively．Enzyme-linked immunosorbent assay（ELISA） and fluorescence scanning were employed to estimate the antigen recognition bioactivity and fluorescence features of recombinant fusion proteins．The half inhibitory concentrations（IC,50,） of the recombinant proteins G8，G8－GFP，and DiG8－GFP were 12.10，14.10 and 2.19 ng/mL，respectively．The results showed that the IC,50, of the monomeric G8－GFP was similar to the non-fused G8，wheras the IC,50, of the divalent DiG8－GFP was improved 5.5 times higher than that of G8．Both G8－GFP and DiG8－GFP emitted fluorescence intensity above 3 000 at the concentration of 0.7 mg/mL，and the DiG8－GFP showed better performance．Indirect ELISA showed that the trimeric recombinant protein TriG8－GFP could bound to AFB,1,－BSA．However，the IC,50, cannot be calculated using the TriG8－GFP as its fluorescence intensity was only 1 700．This study lays a foundation for the subsequent establishment of immunological detection methods based on fluorescence signals，and also provides a reference for the optimization of affinity activity of nanobodies in immunological detection．