α2δ proteins serve as auxiliary subunits of voltage-gated calcium channels (CaV) and regulate the formation and differentiation of synapses. To comparatively study the neuronal localization of the four α2δ isoforms (α2δ-1 to -4), we previously established hemagglutinin (HA) tagging, which allows the surface detection with anti-HA antibody labeling. However, localizing α2δ proteins in synaptic membranes is limited because the synaptic cleft is densely packed with trans-synaptic and extracellular matrix proteins. Thus, classical antibodies have restricted access to extracellular epitopes. Here, we employ an ALFA epitope tag in combination with small camelid antibodies, termed nanobodies, to detect α2δ proteins. We cloned a single ALFA-tag into an extracellular position of α2δ-2 and use recombinant anti-ALFA nanobodies fused to mCherry for immunolabeling. Fluorescent microscopy of cultured hippocampal neurons transfected with double-tagged (HA-tag and ALFA-tag) α2δ-2 reveals a highly specific surface detection with both, antibody and nanobody labeling. Anti-HA antibody labeling shows a dotted expression, which resembles the somato-dendritic localization of L-type calcium channels. In contrast, anti-ALFA nanobody labeling reveals a diffuse expression throughout the plasma membrane, suggesting considerably higher detection sensitivity of α2δ-2 proteins outside channel clusters. To test this hypothesis, we reduced the amount of transfected DNA to limit the availability of α2δ-2 at the cell surface. Here anti-HA antibody and anti-ALFA nanobody labeling showed a similar dotted surface expression pattern. Together this demonstrates the superior detection sensitivity of nanobody labeling of neuronal α2δ proteins which will next be exploited to study the localization of α2δ isoforms in pre- and postsynaptic membranes.