Laccases are multi copper oxidases having wide substrate specificity for phenolic compounds, amines, inorganic compounds like iodine, using oxygen and producing water as by product. The aim of this study is to produce, characterize and purify laccase from yeast isolated from different samples. Yeast isolates were screened for laccase production using tannic acid, catechol, hydroquinone and acetaminophen. Two isolates from soil were selected for further study based on their positive response to screening compounds. Maximum laccase production was achieved with glucose as the best carbon source (10g/l) for Kluyveromyces sp.Dw1 and maltose for Pichia sp.Dw2; Sodium nitrate (3g/l) as the best nitrogen source, optimum pH of 6.0, optimum temperature of 30°C and 35°C for Kluyveromyces sp.Dw1 and Pichia sp.Dw2 respectively. Rice bran supported the highest laccase production of 88.625U/L in Kluyveromyces sp.Dw1 and cane bagasse (79.107U/L) in Pichia sp.Dw2. The addition of inducers like copper sulphate and acetaminophen stimulated maximum laccase production. Maximum activity recorded at pH 9.0, 30°C and 50°C temperature for Kluyveromyces sp.Dw1 and Pichia sp.Dw2 respectively. Laccase activity was retained in the presence of Ca²⁺, K⁺, Hg⁺, EDTA and Benzoic acid. Kluyveromyces sp.Dw1 had K_m value of 0.08mM and 0.13mM for Pichia sp.Dw2 using ABTS as substrate. From this study, Kluyveromyces dobzhanskii Dw1 and Pichia manshurica Dw2 produced laccase that oxidized ABTS with higher laccase production using agro wastes and inducers.