There are no assays for detectingB. burgdorferiantigen in blood of infected Lyme disease individuals. Here, we provide proof-of-principle evidence that we can quantifyB. burgdorferiantigen in spiked blood using a portable smartphone-based fluorescence microscope that measures immunoagglutination on a paper microfluidic chip. We targetedB. burgdorferiOspA to develop a working prototype and added examples of two antigens (OspC and VlsE) that have diagnostic value for discrimination of Lyme disease stage. Using an extensively validated monoclonal antibody to OspA (LA-2), detection of OspA antigen had a broad linear range up to 100 pg/mL in 1% blood and the limit of detection (LOD) was 100 fg/mL (= 10 pg/mL in undiluted blood), which was 1000 times lower than our target of 10 ng/mL. Analysis of the two other targets was done using polyclonal and monoclonal antibodies. OspC antigen was detected at LOD 100 pg/mL (= 10 ng/mL of undiluted blood) and VlsE antigen was detected at LOD 1–10 pg/mL (= 0.1–1 ng/mL of undiluted blood). The method is accurate and was performed in 20 min from sample to answer. When optimized for detecting severalB. burgdorferiantigens, this assay may differentiate active from past infections and facilitate diagnosis of Lyme disease in the initial weeks of infection, when antibody presence is typically below the threshold to be detected by serologic methods.