Hyaluronidase is a protein whose enzymatic activity is successfully employed in extravasation therapy. Taking into account that several proteins (e.g. gelatin and albumin) have been employed as natural polymers for the preparation of microspheres, this work approaches a comprehensive investigation on hyaluronidase injectable microparticles. The goals are either to obtain a sustained release preparation of hyaluronidase or to use the enzyme as drug carrier. Microspheres have been prepared using a water-in-oil emulsification technique, and they have been crosslinked either by thermal or chemical means. Results show that hyaluronidase microspheres with good morphological characteristics can be obtained by this preparation method. Manufacturing variables influence the enzymatic activity of the microspheres, which can be highly preserved under mild experimental conditions. Moreover, the suitability of this enzyme as a microparticulate drug carrier has been shown by the successful encapsulation of hydrocortisone sodium succinate.