The present study investigated the effects of multiple sperm collections on sperm quality, seminal plasma ionic and biochemical parameters, and the relationship between measured variables in the Bulatmai barbel ( Luciobarbus capito ) during the breeding season. For this purpose, the sperm of males ( n = 10) with a mean body weight of 1761.5 ± 385.3 g and an average total length of 55.6 ± 2.8 cm were collected through the first and second stripping process at a 1‐week interval. According to the results, sperm quality parameters, including sperm volume (SV), spermatocrit, spermatozoa concentration, total sperm production (TSP), and total motility (%) significantly decreased at the second sperm sampling ( p < 0.05). The seminal plasma from the first sperm sampling had higher cholesterol and sodium levels ( p < 0.05). Besides, the osmolality of the first stripping was notably higher than comparison of the second stripping ( p < 0.05). The first and second strippings revealed significant correlations among SV–TSP, spermatocrit–TSP, spermatocrit–spermatozoa concentration, and TSP–spermatozoa concentration. Unique correlations include motility percentage–motility duration (MD), glucose‐calcium (negative), glucose–MD, cholesterol–potassium, and triglyceride–magnesium in the first stripping and SV–MD, SV–spermatozoa concentration, spermatocrit–MD, TSP–MD, and total protein‐osmolality in the second. The results of this study recommend that a single stripping method could be more suitable for controlled reproduction programs of Bulatmai barbel males at the hatchery centers to ensure optimal sperm viability and enhance reproductive success.