The use of ultrasonography as a noninvasive tool for assessing the reproductive status of the male Yangtze finless porpoise (YFP; Neophocaena phocaenoides asiaeorientalis) was validated by correlating ultrasonographically determined testicular volume (TV) and testicular parenchyma pixel intensity (PI) with serum testosterone (T) concentration. The testes of 13 free-ranging male YFPs from the Tian-e-Zhou Reserve and three captive animals from the Baiji Dolphinarium (Wuhan, China) were examined ultrasonographically during April 2008. Testis volume was determined using Lambert’s formula for an ellipsoid. Testicular parenchyma PI was evaluated by analyzing testicular ultrasonograms using pixel analysis software (Image J). Serum T concentrations were determined using a single-antibody radioimmunoassay. The TV, PI, and serum T concentration were low and similar in animals with body length <133 cm, highest in those with body length ≥142 cm, and highly variable in those with body length from 133 to 141 cm. Both TV and PI were closely correlated with serum T concentration (r = 0.91 and r = 0.85, respectively; P < 0.01), indicating a consistent association between structural and functional development of the testis. In conclusion, we inferred that puberty onset in male YFPs occurred when TV was >150 cm3 and PI was >60 during the breeding season and that testicular ultrasonography and pixel analysis was an efficient, noninvasive, real-time tool to evaluate testicular function of live male YFPs.