Biological Hotspot research of fish larvae in estuarine waters of Timbulsoko Village, Demak was conducted in April-June 2019 in Timbulsoko Village. Timbulsloko has fertile waters because many fishermen make this location as a fishing ground area. Timbulsloko has the potential to become a nursery ground area and feeding ground for fish due to natural mangrove habitat but the abrasion disaster resulted in the degradation of the nursery ground habitat for early stage fish. The results showed that fish larvae caught in the waters of Timbulsloko Village consisted of 13 families. The composition of the types of fish larvae caught are Mugilidae, Siganidae, Gobiidae, Leiognathidae, Scatophagidae, Chanidae, Latidae, Engraulidae, Gerreidae, Carangidae, Bagridae, Sillaginidae, Ambassidae. The most common types of fish larvae are Ambassidae fish larvae, which are 46.98%, while the least caught are Carangidae fish larvae, Sillaginidae which is 1.01%. The largest abundance of fish larvae is 428,271 ind / m3 found at D2P2, while the abundance of fish larvae is at least at E1P1270 point with an abundance value of 51,498 ind / m3. The similarity of ecological habitat values at D2P2 and A2G1 points based on PCA analysis and the similarity of contours from spatial depth interpolation indicate the biological hotspot in the mangrove waters of Timbulsloko Village.