Abstract Alkaline phosphatases producing microbes are wide spread in nature; ALP is a hydrolase enzyme functioning at alkaline pH, able to hydrolyze phosphates from many types of molecules; this initiating many applications. Experimental designs were applied to evaluate the culture conditions affecting ALP production by Geobacillus thermodenitrificans strain I2, where glucose, yeast-extract and agitation were the most significant variables according to Plackett-Burman design (PBD). The optimum levels of these variables were assessed through Box–Behnken design (BBD) to be (g/L): glucose 26.4, yeast-extract 29.72 and agitation 150rpm, with a predicted enzyme activity of 8.149U/ml. The measured activity upon application the optimized medium was 7.896U/ml. Enzyme was partially purified (42%; 12 fold) through precipitation by ammonium sulfate and anion column, with two protein bands (>70 & ~40kDa) in SDS-PAGE. A partial gene translated sequence proved that the natively expressed ALP by studied strain is related to the band with ~40kDa. The partially purified enzyme exhibited maximum activity at 60°C and pH9.0, with remarkable temperature stability up to 65°C. The results indicated an enhancement in enzyme activity with metals (Mg2+, Mn2+) and reducing agents (DTT & s-mercaptoethanol) whereas, other metals (Zn2+, Hg2+), and anionic surfactant (SDS); EDTA; and PMSF inhibited the enzyme activity. The dependence on enzyme and substrate concentration was represented in Lineweaver-Burk plot where, Km and Vmax were 40mM and 25U/mg protein, respectively. In conclusion, the promising traits of studied ALP would underpin its efficient exploitation in several industries to cope with demands of worldwide enzyme markets.