Pemotongan hewan di Rumah Potong Hewan (RPH) merupakan salah satu tahap terpenting dalam rantai penyediaan daging sapi di Indonesia. Kualitas daging yang dihasilkan di RPH terpengaruh dari proses penerapan higiene dan sanitasi. Praktik higiene dan sanitasi yang buruk di lingkungan RPH dapat menjadi sumber pencemaran mikroorganisme terhadap daging yang dihasilkan serta lingkungan sekitar rumah potong hewan. Beberapa tahun terakhir telah terjadi peningkatan jumlah bakteri Enterobacteriaceae yang menghasilkan extended spectrum β-lactamase. ESBL merupakan enzim yang menginaktivasi sebagian besar antibiotik golongan β-laktam, termasuk extended-spectrum dan very-broad-spectrum sefalosporin dan monobaktam. Terkait dengan persoalan tersebut maka dilakukan penelitian ini yang bertujuan untuk mengetahui ada tidaknya Enterobacteriaceae penghasil ESBL pada feses sapi di Rumah Potong Hewan. Sampel penelitian berjumlah 11 sampel feses sapi yang diambil dari RPH. Penelitian dilakukan dengan cara isolasi bakteri Enterobacteriaceae dengan metode Quadrant Streak, selanjutnya dilakukan identifikasi bakteri dengan pewarnaan Gram. Uji konfirmasi produksi ESBL dilakukan dengan metode difusi cakram. Hasil penelitian menunjukkan seluruh sampel dinyatakan negatif terhadap Enterobacteriaceae penghasil ESBL, hal ini didasari oleh zona hambat yang terbentuk pada antibiotik seftazidim dan sefotaksim diatas >23mm. Dengan demikian dapat disimpulkan bahwa tidak terdapat Enterobacteriaceae penghasil ESBL pada feses sapi di Rumah Potong Hewan. Kata kunci: Enterobacteriaceae, rumah potong hewan,, extended spectrum β-lactamase dan feses sapi Slaughtering animals in Slaughterhouses (RPH) is one of the most important stages in the beef supply chain in Indonesia. The quality of meat produced in slaughterhouses is affected by the process of applying hygiene and sanitation. Poor hygiene and sanitation practices in the RPH environment can be a source of microorganism pollution to the meat produced and the environment around slaughterhouses. In recent years there has been an increase in the number of Enterobacteriaceae bacteria which produce an extended spectrum of β-lactamase.. An increasing number of Enterobacteriaceae bacteria that produce extended spectrum β-lactamase (ESBL) have occurred over the past few years. Extended Spectrum β-Lactamase is an enzyme that activates most β-lactam antibiotics, including extended-spectrum and very-broad-spectrum cephalosporins and monobactams. Related to this problem, this study was conducted which aims to determine the presence or absence of ESBL producing Enterobacteriaceae from cattle feces in slaughterhouse. The research sample consisted of 11 samples cattle feses taken from slaughterhouse. The study was conducted by isolation of Enterobacteriaceae bacteria by the Quadrant Streak method, then identification of bacteria by Gram staining was carried out. The ESBL production confirmation test was carried out using the disc diffusion method. The results showed that all samples were negative of Enterobacteriaceae producing ESBL, this was based on inhibition zones formed on ceftazidim and cefotaxim antibiotics above > 23mm. Thus it can be concluded that there are no Enterobacteriaceae producing ESBL from cattle feces in slaughterhouse. Keywords: Enterobacteriaceae, slaughterhause, extended spectrum β-lactamase and cattle feses