Circulating hormones, that mediate communications across organs to maintain physiological balance, are commonly detected and quantified using enzyme-linked immunosorbent assays (ELISAs). However, while ELISA is well suited for organisms where sample blood can be readily obtained, its application is considerably more challenging in smaller organisms, particularly Drosophila, which has gained widespread use in recent years for physiological studies. Here, we present sensitive phage display–mediated immuno-PCR (PD-iPCR) to detect Drosophila hemolymph proteins via two approaches: 1) by identifying high-affinity nanobodies through phage display library screening and subsequent affinity maturation and 2) by generating a knock-in fly line producing secreted proteins tagged with tandem NanoTags composed of VHH05 and 127D01. Using these approaches, we successfully established PD-iPCR to detect insulin-binding ImpL2 protein in fly hemolymph. Notably, the tandem NanoTag-based sandwich PD-iPCR enabled highly sensitive detection of tagged antigens, allowing us to quantify elevated ImpL2 levels in the hemolymph of starved flies and those bearing Yki-induced gut tumors. Collectively, our results demonstrate that PD-iPCR enables detection of endogenous, low-abundance circulating hormones in Drosophila, providing a powerful tool for studying interorgan communication.