A novel nanofiber composite nanofiltration membrane was fabricated through coating calcium alginate hydrogel on electrospun polyhydroxybutyrate/carbon nanotubes (CaAlg-c-PHB/CNT) nanofiber membrane. The CaAlg-c-PHB/CNT nanofiber composite membrane was investigated by means of SEM, FTIR, Raman, XRD, DSC, TGA, zeta potential and water contact angle (WCA) measurements. The tensile tests of the CaAlg-c-PHB/CNT nanofiber composite membrane were performed in dry and wet state. The filtration properties of the CaAlg-c-PHB/CNT nanofiber composite membrane were measured in term of flux and rejection rate at operation pressure ranged from 0.1 to 0.7 MPa. The antifouling properties were investigated by emulsified oil-water and BSA solution separation. Results showed that the CaAlg-c-PHB/CNT nanofiber composite membrane with 1 wt% CNT had the highest tensile strength and initial Modulus with the value of 6.14, 5.12 and 142.64, 127.5 MPa in dry and wet state, respectively. The water contact angle was decreased remarkably from 83.6 degrees to 17.8 degrees after calcium alginate hydrogel coating. The pure water flux (PWF) was increased from 68.61 to 150.72 L/m(2) h with operation pressure increased from 0.1 to 0.7 MPa. Five dyes with different molecular weight ranged from 400 to 900 g/mol were selected for filtration test. The rejection rate could maintain over 90% for dyes with the molecular weight higher than 600 g/mol under 0.5 MPa. The CaAlg-c-PHB/CNT nanofiber composite membrane showed good antifouling performance for both oil and protein. These results indicated that the CaAlg-c-PHB/CNT composite nanofiber membrane can be used as an effective nanofiltration membrane operated at high pressure for dye removal.