Among different pectinases, pectin lyase seems to be the only pectic enzyme capable of breaking down pectin with a high degree of esterification. Trichoderma harziamun is excellent pectinases producer, therefore different isolates were screened by quantitative and qualitative methods. Screening results showed that two isolates showed maximum pectin lyase. Different fermentation conditions like fermentation rate, inoculum size, concentration of peptone, concentration of yeast extract, pH and tween -80 were optimized by different experiments for production of pectin lyase by T. harzianum isolates. Pectin lyase was partially purified by ammonium sulphate precipitation. The fraction, which contained a large amount of pectin lyase was dialyased against 0.2 M phosphate buffer at pH 5.5 for 12 hours and used for SDS-PAGE and characterization of pectin lyase. The partial purified pectin lyase (PL) from T140 and T 325 isolates had a molecular weight of 22 kDa. Purified enzyme form isolates were also characterized at different pH temperature and substrate range. Results were discussed on the basis of to statistical analysis of variance (ANOVA) under Complete Randomized Design (CRD).