Staphylococcus aureus originating from meat is a leading causes of foodborne illness and the presence and spreading of antimicrobial resistant S. aureus is one of the great global public health issue especially in developing countries like Ethiopia. A cross-sectional study was conducted from October 2019 to April 2020 for Identification and Testing Antimicrobial Susceptibility of Staphylococcus aureus isolates from Beef at Municipal Abattoir and Butcher Shops in Dangila Town, Northwest Ethiopia. A swab samples were collected from a municipal abattoir and randomly selected 16 butcher shops (carcasses, knives, cutting boards and hooks). Isolates were identified by the cultural, morphological and biochemical tests. Enumeration of S. aureus were conducted by colony counter and expressed as mean value log10cfu/cm2. Antimicrobial susceptibility testing was carried out according to CLSI 2019 recommendations. Hygienic status of meat handling at abattoir and butcher shops were assessed by interview question and observation. From a total of 128 swab samples collected from a municipal abattoir and selected butcher shops, 48.43%) (31/64) and 54.68% (35/64) were found positive for S. aureus, respectively with overall positive rate of 51.56% (66/128). Microbial load and mean bacterial count analyses of S. aureus isolated from swaps of carcasses, knives, cutting tables and hooks at abattoir and selected butcher shops were computed and found to be 5.5, 8.2, 7.5, 5.7log10cfu/cm2 and 5.7, 7.2, 6.7 and 5.9log10cfu/cm2, respectively. The antimicrobial susceptibility testing conducted on 66 isolated S. aureus using the disc diffusion method on Muller Hinton agar showed 100% of S. aureus isolates to be resistant to at least one antimicrobial compound, and 39.4% were multi drug resistant (MDR). Resistance to methicillin was 87.9% followed by Tetracycline (18.2%), Gentamycin (12.1%) and Sulfonamide (12.1%). In this study, S. aureus was found the major contaminant of beef value chain from abattoir to butcher shops. The overall higher loads of the S. aureus occurs on the beef at the butcher shops relative to abattoir due to further contaminations during transportation and handling at the retail houses. Most of the isolates were resistant to single to multiple antimicrobials. Therefore, hygienic meat processing should be implemented in order to prevent S. aureus intoxication, and misuse and over use of antimicrobial agents should be controlled. Keywords: Abattoir, Antimicrobial, Beef, Butcher shop, Dangila, Staphylococcus aureus