Purpose To investigate the microstructure of the lacrimal canaliculus and the characteristics of lacrimal canalicular diseases by 80-MHz ultrasound biomicroscopy (UBM). Methods This study included 33 participants: 20 normal subjects (40 eyes), 2 patients with chronic lacrimal canaliculitis (4 eyes), 10 patients with chronic dacryocystitis (16 eyes), and 1 patient with lacrimal punctum atresia (2 eyes). All participants underwent 80-MHz UBM; disease-specific features were noted. Results On 80-MHz UBM of the lacrimal canaliculi (vertical section) in normal subjects, low echo of the lacrimal canalicular lumen and high echo of the lacrimal canalicular wall were observed. The uniform low echo near the wall was the mucosal epithelium. The outermost layer of medium-to-high echo was the subepithelial elastic fibrous layer. In the horizontal section, the lumen was continuous. Two linear high echoes parallel to the canalicular wall could be observed at the center of the lacrimal canaliculus, which were sometimes attached and sometimes separated. When separated, the center of the lacrimal canaliculus was a low echo area (lumen). Lacrimal canaliculitis (vertical section) showed obvious ectasia of the lacrimal canalicular lumen, with a high echo mass shadow, which might have been calculi, and uneven thickness of the mucosal epithelium with a slightly high echo shadow. In the horizontal section, the lumen varied in size with clear boundaries of medium and high echoes. The central linear high echoes of the lumen were absent, and the echoes of the mucosal epithelium were discontinuous. In chronic dacryocystitis, the lacrimal canalicular lumen was extensively enlarged, with continuous echoes and uniform thickness of the mucosal epithelium and homogeneous patches of slightly higher echoes. Lacrimal punctum atresia indicated that the lacrimal canaliculus existed in both eyes and its structure was normal. Conclusions The 80-MHz UBM is a new non-invasive technique that can be used for clear visualization of the fine structure of the lacrimal canaliculus, including the mucosal epithelium and subepithelial elastic fiber layer. The use of this approach will improve understanding of the hierarchical structure of the lacrimal canaliculi and provide a comprehensive basis for diagnosis, differential diagnosis, and treatment plan in patients with lacrimal passage diseases.