The aim of this study was to evaluate the effects of smoking on surgical outcome and hearing results in tympanoplasty by serum cotinine analysis as an objective method. Furthermore, type of grafting for the smoking patients has been investigated in a prospective cohort in a tertiary medical center. The overall success rate of long-term surgical outcome was 70.1% out of 77 tympanoplasty operations. On the basis of cotinine enzyme immunoassay, 56 patients had a serum cotinine measurement below 17.5 ng/ml and 21 patients above that. While the graft take rate in the non-smoking group was 76.8%, it was 52.4% in the smoking group and this difference was statistically significant (p = 0.037). While mean graft take rate in the temporalis fascia group was 25% for smokers, mean graft take rate in cartilage shield tympanoplasty group it was 88.9%, and for smokers it was 52.4%. Cotinine is a major metabolite of nicotine and is a reliable marker to differentiate smoking patients from non-smokers. Smoking status was found as a significant prognostic factor influencing the success rate of tympanoplasty negatively and the influence of a more stable grafting technique was demonstrated on smoking patients undergoing tympanoplasty procedure.