The effect of inclusion complex formation on the fluorescence properties of berberine, a clinically important natural alkaloid, was studied using cucurbit[7]uril as macrocyclic host compound. The formation of a very stable 1:1 inclusion complex led to about 500-fold fluorescence intensity enhancement, which facilitated the detection of berberine even below nanomolar concentration. Addition of NaCl caused a significant change in the association constant and the fluorescence characteristics of the complex, whereas the variation of the anion had a small effect. 1-Alkyl-3-methylimidazolium type ionic liquids altered the fluorescent properties of the berberine−cucurbit[7]uril complex much more efficiently than did NaCl. Time-resolved fluorescence studies showed ternary complex formation. Because berberine fluorescence is insensitive to pH and increases substantially upon inclusion in cucurbit[7]uril, the reversible self-assembly of this host−guest pair may find analytical application in enzyme assays.