New methods are needed to modify silk biomaterials with bioactive molecules for tissue engineering and drug delivery. In the present study, silk fibroin in solution or in microsphere format was coupled with NeutrAvidin via carbodiimide chemistry. Silk fibroin retained its self-assembly features after reaction. It was found that more than four NeutrAvidin molecules bound to one silk molecule. Non-specific binding of biotin or NeutrAvidin to silk microspheres could be reduced by pre-treatment of the microspheres with BSA or post-treatment with detergent. The NeutrAvidin-coupled silk microspheres were coupled with biotinylated anti-CD3 antibody and the functionalized microspheres were able to specifically bind to the CD3 positive T-lymphocytic cell line Jurkat.