Gracllaria sp. merupakan rumput laut yang memiliki nilai ekonomis tinggi karena menghasilkan agar. Agar banyak dimanfaatkan dalam bidang industri maupun pangan. Kualitas Gracilaria sp. ditentukan oleh kadar agar, pigmen serta proksimatnya. Penelitian ini bertujuan untuk mengkaji pengaruh parameter kualitas media tumbuh Gracilaria sp. terhadap biosintesis agar, klorofil a, karoten dan kadar proksimat (protein, karbohidrat, lemak, abu, dan air). Lokasi pengambilan sampel di reservoir dan biofilter tambak udang Vaname (Litopenaeus vannamei). Kondisi lingkungan perairan yang diukur adalah kandungan nitrat dan fosfat, suhu air, salinitas, DO (oksigen terlarut), serta pH (Derajat keasaman). Ekstraksi agar dilakukan dengan metode alkali. Karakterisasi agar menggunakan analisis Fourier Transform Infra Red. Hasil penelitian menunjukkan kandungan agar (31,4 ± 0,42%) pada reservoir lebih baik dibandingkan biofilter. Begitu pula dengan konsentrasi klorofil a (19,61 ± 0,04 mg/g), karotenoid (7,42 ± 0,21 µmol/g) serta kadar protein (15,38 ± 0,27%). Kadar lemak, karbohidrat, air dan abu pada kedua lokasi tidak berbeda nyata. Hasil karakterisasi agar menujukkan keberadaan gugus galaktosa 3,6-anhydro-L-galaktosa. Gracilaria sp. is well known as to has a high economically value, due to the application of the agar content that is used in various industries especially in food industry. The quality of Gracilaria sp. depends on agar and pigment content as well as proximate analysis. This study aimed to investigate the effect of water quality parameter on Gracilaria sp habitats to agar, chlorophyl a, carotene and proximate analysis (protein, carbohydrate, total lipid, ash, water content). Samples were collected from reservoir and biofilter from Litopenaeus vannamei’s waste pond. Water quality parameter measured were nitrate and phosphate, water temperature, salinity, dissolved oxygen (DO) and pH. Agar extraction was done by alkali methods. Agar and its chemical structure were characterized by FT-IR analysis. Results showed that agar content (31.4 ± 0.42%) on reservoir was better than biofilter. Higher results from reservoir samples were also found in chlorophyl a (19.61 ± 0.04 mg/g), carotenoids (7.42 ± 0.21 µmol/g) and protein level (15.38 ± 0.27 %). On the other hand, total lipid, carbohydrate, water and ash content were similar. Based on FT-IR analysis shows that the galactose 3,6-anhydro-L-galactose was present.