Alvimopan (ALV), a medication for the treatment of postoperative ileus, is a biopharmaceutical classification system (BCS) class IV drug whose poor aqueous solubility restricts its bioavailability. The aim of the research was to create nanocrystals that would help ALV dissolve more quickly and be more bioavailable when taken orally. To improve oral bioavailability, the ALV nanocrystals (ALV-NC) were made using a wet media milling technique and fluidized bed processing. In, formulation screening, sodium lauryl sulphate (SLS) was used as an electrostatic stabilizer with hydroxypropyl methylcellulose (HPMC) as a steric stabilizer and sucrose to suppress the hydration of the polymer matrix to accelerate drug release using the wet-milling method. The formulated ALV-NC showed a small and uniform particle size with a marked increase in dissolution in different dissolution media compared with the marketed product (Entereg®). The formulation and preparation processes were further refined by a 32 factorial design with SDS content (X1) and HPMC content (X2) as independent factors and particle size, in-vitro release (IVR) at 30 min., and zeta potential as variables for responses. The optimal condition was determined as a combination of 1.07 mg per capsule of SLS and 0.31 mg per capsule of HPMC. The particle size (D 90), IVR at 30 min., and zeta potential were all determined to be (Y1) 472 ±12.57 nm, (Y2) 100.4 %, and (Y3) -39.1 mV, respectively. The optimized formulation showed irregular shape morphology as observed by scanning electron microscopy (SEM) and crystallinity, along with molecular interaction between drug and stabilizer determined by differential scanning calorimetry (DSC), X-ray powder diffraction (PXRD), and Fourier transform infrared spectroscopy (FT-IR). Furthermore, the in vivo pharmacokinetics of the formulated ALV-NC were evaluated in Sprague-Dawley rats by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The results indicated that the Cmax and AUC0-24 of ALV-NC were 2.38-fold and 1.74-fold higher than those of the marketed capsule formulation (Entereg®), respectively. The screening and formulation optimization procedures used in this study are a practically viable method for increasing the oral bioavailability of ALV.