The Mona Lisa's ambiguous expression has captivated viewers for centuries, prompting diverse explanations. This article proposes a novel interpretation grounded in the psychological theory of perceptual organisation. Central to the investigation is the “Ambiguity Smudge”, a dark region above the mouth, hypothesised to influence perceived expression due to perceptual organization. Through carefully crafted artwork and systematic manipulations of Mona Lisa reproductions, experiments reveal how alterations of the Ambiguity Smudge generate distinct expressions. Specifically, the manipulation of the perceptual relationships between the Ambiguity Smudge and the mouth yields significant shifts in perceived expression. These findings not only underscore the pivotal role of psychological principles in shaping ambiguous expressions in the Mona Lisa, but also extend to other Leonardo’s portraits, namely La Bella Principessa and Scapigliata. This study sheds light on the intersection of psychology and art, offering new perspectives on timeless masterpieces.