Purpose <p>This study aimed to explore the roles of spermatozoal sialidase NEU1 and NEU3 in affecting the fertilization rate of in vitro fertilization (IVF), and whether sperm sialidase NEU1 and NEU3 can be used as predictors of IVF outcome.</p> Material and methods <p>This was a prospective cohort study that collected semen samples from 194 IVF couples between January 2024 and April 2024. Patients were grouped based on oocyte maturity and routine semen analysis. Detection of sperm NEU1 and NEU3 was carried out in semen from the same ejaculate for IVF by using flow cytometry, followed by correlation analysis with fertilization rates.</p> Results <p>NEU1 and NEU3 are independent indicators separate from regular semen parameters. With a metaphase II (MII) rate ≥80% and normal semen routine analysis, sperm NEU1 (<i>P</i> = 0.0300) and NEU3 (<i>P</i> = 0.0253) levels were significantly different demarcated by 80% fertilization rate. Lower NEU1 and NEU3 expression on sperm is associated with lower fertilization rates. ROC curve analyses displaying the diagnostic accuracy of NEU1 (<i>P</i> = 0.0306, AUC = 0.660) and NEU3 (<i>P</i> = 0.0259, AUC = 0.665). <i>χ</i>² test analyzes the fertilization rate and the different cut-off rate for NEU1 (<i>χ</i>² = 4.671, <i>P</i> = 0.0307) or NEU3 (<i>χ</i>² = 6.377, <i>P</i> = 0.0116).</p> Conclusions <p>NEU1 and NEU3 on sperm can predict the IVF fertilization outcome in the group with oocyte MII rate ≥80%. The protein levels of NEU1 and NEU3 on sperm could be a new index for assisted reproductive technology (ART).</p>