The use of "one-shot" CO laser technique for a primary small-fenestra stapedotomy is well established, but few papers report the long-term functional results. We retrospectively reviewed medical records of 198 patients, treated for otosclerosis from January 2008 to December 2011, at the Department of Head and Neck Surgery, Catholic University of Rome. Statistical comparison between audiological thresholds obtained 24 h preoperatively, at early (4 weeks) and late postoperative examinations (mean time 45 months), was performed. Comparison of preoperative vs both early and late postoperative ACPTA showed a statistically significant difference (respectively 55 vs 33 and 31 dB; p < 0.001). No statistical difference was observed between preoperative, early and late postoperative BCPTA (respectively 23 vs 23 and 22 dB; p > 0.05). Both early and late postoperative ABG improved significantly compared to the preoperative one (respectively 10 and 9 vs 32 dB; p < 0.001). No statistical difference was found in comparison of early vs late postoperative ACPTA (respectively 33 vs 31 dB; p > 0.05), early vs late postoperative ABG (respectively 10 vs 9 dB; p > 0.05) and early vs late ABG gain (respectively 22 vs 23 dB; p > 0.05). No subjects developed postoperative complications requiring revision surgery or late deterioration of hearing threshold. The analysis of our data suggests that "one-shot" CO laser stapedotomy is an effective and safe procedure: it allows a rapid stapedotomy without damages for the inner ear and optimal functional results that remain stable during the years.