The　biochemical　properties　of　all　four　β-galactosidases　from　Aspergillus　niger　strain　F0215　were　investigated.　The　genes　encoded　β-galactosidases　were　cloned　and　expressed　in　Pichia　pastoris　GS115.　The　recombinants　GS115　(PIC-bgl　A),　GS115　(p　PIC-bgl　B),　GS115　(p　PIC-bgl　C)　and　GS115　(p　PIC-bgl　E)　were　subsequently　constructed　and　the　recombinant　β-galactosidases　BglA,　BglB,　BglC　and　BglE　were　prepared　by　using　shaking　flask　fermentation　and　methanol　induction　followed　by　comprehensive　investigation　of　their　biochemical　properties.　The　temperature　optimum　of　BglA,　BglB,　BglC　or　BglE　was　60,　50,　60　and　50　,　respectively,　the　p　H　optima　were　of　4.0,　4.5,　3.5　and　4.5,　respectively.　Their　activities　were　enhanced　by　Mg2+,　Ca2+,　Na+or　Mn2+but　significantly　inhibited　by　Cu2+,　Fe2+,　Ni2+,　Zn2,　Li+or　EDTA.　The　Km　value　of　BglA,　BglB,　BglC　or　BglE　towards　ONPG　was　determined　to　be　0.43,　0.22,　0.37　mmol/L　and　0.46　mmol/L.　BglA,　BglB,　BglC　and　BglE　performed　a　specificity　towards　lactose　as　sole　substrate　with　the　activities　of　hydrolysis　and　transglycosylation.　All　identified　four　β-galactosidases　possessed　a　typical　molecular　structure　of　fungalβ-galactosidases.　β-Galactosidase　BglD　was　not　existed　in　the　test　strain　although　it　was　predicted　in　the　reference　genome　sequence.　©　2017,　Editorial　Office　of　Journal　of　CIFST.　All　right　reserved.